Laboratory of Cellular and Tissue Engineering

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2017

165. Fong, L.S.E., and Yu, H. (2017) Organs-on-chips: Filtration enabled by differentiation. Nature Biomedical Engineering, 10 May 2017; 1(5):0074. DOI: 10.1038/s41551-017-0074

The efficient generation of mature podocytes from induced pluripotent stem cells makes possible the recapitulation of renal blood filtration on a chip.

164. Fang, Y., Zhuo, S., Qu, Y., Choudhury, D., Wang, Z., Iliescu, C., and Yu, H. (2017) On Chip Two-photon Metabolic Imaging for Drug Toxicity Testing. Biomicrofluidics, May 2017; 11(3): 034108. DOI: 10.1063/1.4983615

We have developed a microfluidic system suitable to be incorporated with a metabolic imaging method to monitor the drug response of cells cultured on a chip. The cells were perfusion-cultured to mimic the blood flow in vivo. Label-free optical measurements and imaging of nicotinamide adenine dinucleotide and flavin adenine dinucleotide fluorescence intensity and morphological changes were evaluated noninvasively. Drug responses calculated using redox ratio imaging were compared with the drug toxicity testing results obtained with a traditional well-plate system. We found that our method can accurately monitor the cell viability and drug response and that the IC50 value obtained from imaging analysis was sensitive and comparable with a commonly used cell viability assay: MTS (3–(4,5-dimethylthiazol-2-yl)–5–(3-carboxymethoxyphenyl)-2–(4-sulfo-phenyl)-2H-tetrazolium) assay. Our method could serve as a fast, non-invasive, and reliable way for drug screening and toxicity testing as well as enabling real-time monitoring of in vitro cultured cells.

163. Gupta, K., Li, Q., Fan, J.J., Fong, E.L.S., Song, Z., Mo, S., Tang, H., Ng, I.C., Ng, C.W., Zhuo, S., Dong, C.-Y., Low, B.C., Wee, A., Dan, Y.Y., Kanchanawong, P., So, P., Viasnoff, V., and Yu, H. (2017) Actomyosin Contractility Drives Bile Regurgitation as an Early Response During Obstructive Cholestasis. Journal of Hepatology, 18 March 2017; S0168-8278(17): 30061-30062. DOI: 10.1016/j.jhep.2017.01.026

BACKGROUND & AIMS: A wide range of liver diseases manifest as biliary obstruction, or cholestasis. However, the sequence of molecular events triggered as part of the early hepatocellular homeostatic response in obstructive cholestasis is poorly elucidated. Bile canaliculi are dynamic luminal structures that undergo actomyosin-mediated periodic contractions to propel secreted bile. Additionally, pericanalicular actin is known to accumulate during obstructive cholestasis. Therefore, we hypothesize that the pericanalicular actin cortex undergoes significant remodeling as a regulatory response to obstructive cholestasis.
METHODS: Investigations into the effects of obstructive cholestasis were performed in a bile duct ligated mouse model. To elucidate the role of actomyosin contractility, we used sandwich-cultured hepatocytes transfected with various fluorescently labeled proteins and pharmacological inhibitors of actomyosin contractility.
RESULTS: We report here that actomyosin contractility induces transient deformations along the canalicular membrane, a process we have termed inward blebbing. We show that these membrane intrusions are initiated by local ruptures in the pericanalicular actin cortex; and they typically retract following repair by actin polymerization and actomyosin contraction. However, above a certain osmotic pressure threshold, these inward blebs pinch away from the canalicular membrane into the hepatocyte cytoplasm as large vesicles (2-8 μm). Importantly, we show that these vesicles aid in the regurgitation of bile from the bile canaliculi.
CONCLUSION: Actomyosin contractility induces the formation of bile-regurgitative vesicles, thus serving as an early homeostatic mechanism against increased biliary pressure during cholestasis.
LAY ABSTRACT: Bile canaliculi lumen undergoes cyclic expansion and contraction mediated by bile secretion, and resistance from the surrounding actin bundles. Further expansion due to bile duct blockade leads to the formation of inward blebs and vesicles which carry away excess bile to prevent bile build up in the lumen.

162. Fong, L.S.E., Toh, T.B., Chow, E., and Yu, H. (2017) 3D culture as a clinically relevant model for personalized medicine. SLAS Technology, March 2017; 1: 2472630317697251. DOI: 10.1177/2472630317697251

Advances in understanding many of the fundamental mechanisms of cancer progression have led to the development of molecular targeted therapies. While molecular targeted therapeutics continue to improve the outcome for cancer patients, tumor heterogeneity among patients, as well as intratumoral heterogeneity, limits the efficacy of these drugs to specific patient subtypes, as well as contributes to relapse. Thus, there is a need for a more personalized approach toward drug development and diagnosis that takes into account the diversity of cancer patients, as well as the complex milieu of tumor cells within a single patient. Three-dimensional (3D) culture systems paired with patient-derived xenografts or patient-derived organoids may provide a more clinically relevant system to address issues presented by personalized or precision medical approaches. In this review, we cover the current methods available for applying 3D culture systems toward personalized cancer research and drug development, as well as key challenges that must be addressed in order to fully realize the potential of 3D patient-derived culture systems for cancer drug development. Greater implementation of 3D patient-derived culture systems in the cancer research field should accelerate the development of truly personalized medical therapies for cancer patients.

161. Lou, Y.R., Toh, T.C., Tee, Y.H., and Yu, H. (2017) 25-Hydroxyvitamin D3 induces osteogenic differentiation of human mesenchymal stem cells. Scientific Reports, 17 February 2017; 7: 41238. DOI: 10.1038/srep42816

25-Hydroxyvitamin D3 [25(OH)D3] has recently been found to be an active hormone. Its biological actions are demonstrated in various cell types. 25(OH)D3 deficiency results in failure in bone formation and skeletal deformation. Here, we investigated the effect of 25(OH)D3 on osteogenic differentiation of human mesenchymal stem cells (hMSCs). We also studied the effect of 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3], a metabolite of 25(OH)D3. One of the vitamin D responsive genes, 25(OH)D3-24-hydroxylase (cytochrome P450 family 24 subfamily A member 1) mRNA expression is up-regulated by 25(OH)D3 at 250-500 nM and by 1α,25-(OH)2D3 at 1-10 nM. 25(OH)D3 and 1α,25-(OH)2D3 at a time-dependent manner alter cell morphology towards osteoblast-associated characteristics. The osteogenic markers, alkaline phosphatase, secreted phosphoprotein 1 (osteopontin), and bone gamma-carboxyglutamate protein (osteocalcin) are increased by 25(OH)D3 and 1α,25-(OH)2D3 in a dose-dependent manner. Finally, mineralisation is significantly increased by 25(OH)D3 but not by 1α,25-(OH)2D3. Moreover, we found that hMSCs express very low level of 25(OH)D3-1α-hydroxylase (cytochrome P450 family 27 subfamily B member 1), and there is no detectable 1α,25-(OH)2D3 product. Taken together, our findings provide evidence that 25(OH)D3 at 250-500 nM can induce osteogenic differentiation and that 25(OH)D3 has great potential for cell-based bone tissue engineering.

160. Narmada, B.C., Goh, Y.T., Li, H., Sinha, S., Yu, H., and Cheung, C. (2017) Human stem cell-derived endothelial-hepatic platform for efficacy testing of vascular-protective metabolites from nutraceuticals. Stem Cells Translational Medicine, March 2017; 6(3): 851-863. DOI: 10.5966/sctm.2016-0129

Atherosclerosis underlies many cardiovascular and cerebrovascular diseases. Nutraceuticals are emerging as a therapeutic moiety for restoring vascular health. Unlike small-molecule drugs, the complexity of ingredients in nutraceuticals often confounds evaluation of their efficacy in preclinical evaluation. It is recognized that the liver is a vital organ in processing complex compounds into bioactive metabolites. In this work, we developed a coculture system of human pluripotent stem cell-derived endothelial cells (hPSC-ECs) and human pluripotent stem cell-derived hepatocytes (hPSC-HEPs) for predicting vascular-protective effects of nutraceuticals. To validate our model, two compounds (quercetin and genistein), known to have anti-inflammatory effects on vasculatures, were selected. We found that both quercetin and genistein were ineffective at suppressing inflammatory activation by interleukin-1b owing to limited metabolic activity of hPSC-ECs. Conversely, hPSC-HEPs demonstrated metabolic capacity to break down both nutraceuticals into primary and secondary metabolites. When hPSC-HEPs were cocultured with hPSC-ECs to permit paracrine interactions, the continuous turnover ofmetabolitesmitigated interleukin-1b stimulation on hPSC-ECs.We observed significant reductions in inflammatory gene expressions, nuclear translocation of nuclear factor kB, and interleukin-8 production. Thus, integration of hPSC-HEPs could accurately reproduce systemic effects involved in drug metabolism in vivo to unravel beneficial constituents in nutraceuticals. This physiologically relevant endothelial-hepatic platform would be a great resource in predicting the efficacy of complex nutraceuticals and mechanistic interrogation of vascular-targeting candidate compounds.

159. Choudhury, Y., Toh, Y.-C., Xing, J., Qu, Y., Poh, J., Li, H., Tan, H.S., Kanesvaran, R., Yu, H., and Tan, M.-H (2017) Patient-specific hepatocyte-like cells derived from induced pluripotent stem cells model pazopanib-mediated hepatotoxicity. Scientific Reports, 25 January 2017; 7: 41238. DOI: 10.1038/srep41238

Idiosyncratic drug-induced hepatotoxicity is a major cause of liver damage and drug pipeline failure, and is difficult to study as patient-specific features are not readily incorporated in traditional hepatotoxicity testing approaches using population pooled cell sources. Here we demonstrate the use of patient-specific hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells for modeling idiosyncratic hepatotoxicity to pazopanib (PZ), a tyrosine kinase inhibitor drug associated with significant hepatotoxicity of unknown mechanistic basis. In vitro cytotoxicity assays confirmed that HLCs from patients with clinically identified hepatotoxicity were more sensitive to PZ-induced toxicity than other individuals, while a prototype hepatotoxin acetaminophen was similarly toxic to all HLCs studied. Transcriptional analyses showed that PZ induces oxidative stress (OS) in HLCs in general, but in HLCs from susceptible individuals, PZ causes relative disruption of iron metabolism and higher burden of OS. Our study establishes the first patient-specific HLC-based platform for idiosyncratic hepatotoxicity testing, incorporating multiple potential causative factors and permitting the correlation of transcriptomic and cellular responses to clinical phenotypes. Establishment of patient-specific HLCs with clinical phenotypes representing population variations will be valuable for pharmaceutical drug testing.

158. Domian, I.J., Yu, H., and Mittal, N. (2017) On Materials for Cardiac Tissue Engineering. Advanced Healthcare Materials, January 2017; 6(2): 1600768. DOI: 10.1002/adhm.201600768

In this essay the authors argue that chamber pressure dominates the biomechanics of the contraction cycle of the heart, while tissue stiffness dominates the relaxation cycle. This appears to be an under-recognized challenge in cardiac tissue engineering. Optimal approaches will involve constructing chambers or modulating the stiffness of the scaffold/substrate in synchrony with the beating cycle.

157. Ni, M., Zhuo, S., So, P.T.C., and Yu, H. (2017) Fluorescent Probes for Nanoscopy: Four Categories and Multiple Possibilities. Journal of Biophotonics, January 2017; 10(1): 11-23. DOI: 10.1002/jbio.201600042

Nanoscopy enables breaking down the light diffraction limit and reveals the nanostructures of objects being studied using light. In 2014, three scientists pioneered the development of nanoscopy and won the Nobel Prize in Chemistry. This recognized the achievement of the past twenty years in the field of nanoscopy. However, fluorescent probes used in the field of nanoscopy are still numbered. Here, we review the currently available four categories of probes and existing methods to improve the performance of probes.

2016

156. Song, Z., Shanmugam, M.K., Yu, H., and Sethi G. (2016) Butein and Its Role in Chronic Diseases. Advances in Experimental Medicine and Biology, 25 September 2017; 928: 419-433. DOI: 10.1007/978-3-319-41334-1_17

Natural compounds isolated from various plant sources have been used for therapeutic purpose for centuries. These compounds have been routinely used for the management of various chronic ailments and have gained considerable attention because of their significant efficacy and comparatively low side effects. Butein, a chacolnoid compound that has been isolated from various medicinal plants has exhibited a wide range of beneficial pharmacological effects, such as anti-inflammatory, anticancer, antioxidant, and anti-angiogenic in diverse disease models. This article briefly summarizes the past published literature related to the therapeutic and protective effects of butein, as demonstrated in various models of human chronic diseases. Further analysis of its important cellular targets, toxicity, and pharmacokinetic profile may further significantly expand its therapeutic application.

155. Zhu, L., Xia, H., Wang, Z., Fong, L.S.E., Fan, J., Tong, W.H., Seah, Y.P.D., Zhang, W., Li, Q., and Yu, H. (2016) A vertical-flow Bioreactor Array Compacts Hepatocytes for Enhanced Polarity and Functions. Lab on a Chip, 16(20): 3898-3908. DOI: 10.1039/c6lc00811a

Although hepatocytes in vivo experience intra-abdominal pressure (IAP), pressure is typically not incorporated in hepatocyte culture systems. The cuboidal cell shape and extent of intercellular contact between cultured hepatocytes are critical parameters that influence the differentiated hepatic phenotype. Using a microfluidic device, the application of pressure to artificially compact cells and forge cell–cell interactions was previously demonstrated to be effective in accelerating hepatic repolarization. In seeking to implement this approach to higher throughput culture platforms for potential drug screening applications, we specifically designed a vertical-flow compaction bioreactor array (VCBA) that compacts hepatocytes within the range of IAP and portal pressure in vivo in a multi-well setup. As a result of vertical perfusion-generated forces, hepatocytes not only exhibited accelerated repolarization, an in vivo-like cuboidal morphology, but also better maintained hepatic functions in long-term culture as compared to the same cells cultured under static conditions. As a novel engineering tool to modulate cell compaction and intercellular interactions, this platform is a promising approach to confer tight control over hepatocyte repolarization for in vitro culture.

154. Fong, L.S.E., Harrington, D.A., Farach-Carson, M.C., and Yu, H. (2016) Heralding a New Paradigm in 3D Tumor Modeling. Biomaterials, November 2016; 108: 197-213. DOI: 10.1016/j.biomaterials.2016.08.052

Numerous studies to date have contributed to a paradigm shift in modeling cancer, moving from the traditional two-dimensional culture system to three-dimensional (3D) culture systems for cancer cell culture. This led to the inception of tumor engineering, which has undergone rapid advances over the years. In line with the recognition that tumors are not merely masses of proliferating cancer cells but rather, highly complex tissues consisting of a dynamic extracellular matrix together with stromal, immune and endothelial cells, significant efforts have been made to better recapitulate the tumor microenvironment in 3D. These approaches include the development of engineered matrices and co-cultures to replicate the complexity of tumor-stroma interactions in vitro. However, the tumor engineering and cancer biology fields have traditionally relied heavily on the use of cancer cell lines as a cell source in tumor modeling. While cancer cell lines have contributed to a wealth of knowledge in cancer biology, the use of this cell source is increasingly perceived as a major contributing factor to the dismal failure rate of oncology drugs in drug development. Backing this notion is the increasing evidence that tumors possess intrinsic heterogeneity, which predominantly homogeneous cancer cell lines poorly reflect. Tumor heterogeneity contributes to therapeutic resistance in patients. To overcome this limitation, cancer cell lines are beginning to be replaced by primary tumor cell sources, in the form of patient-derived xenografts and organoids cultures. Moving forward, we propose that further advances in tumor engineering would require that tumor heterogeneity (tumor variants) be taken into consideration together with tumor complexity (tumor-stroma interactions). In this review, we provide a comprehensive overview of what has been achieved in recapitulating tumor complexity, and discuss the importance of incorporating tumor heterogeneity into 3D in vitro tumor models. This work carves out the roadmap for 3D tumor engineering and highlights some of the challenges that need to be addressed as we move forward into the next chapter.

153. Mittal, N., Tasnim, F., Cao, Y., Qu, Y., Phan, D., Choudhury, Y., Tan, M.-H., and Yu, H. (2016) Substrate stiffness modulates the maturation of human pluripotent stem cell derived hepatocytes. ACS Biomaterials Science & Engineering, 2(9): 1649-1657. DOI: 10.1021/acsbiomaterials.6b00475

Obtaining functional hepatocytes from human pluripotent stem cells (hPSCs) holds great potential for applications in drug safety testing, as well in the field of regenerative medicine. However, developing functionally mature hPSC-derived hepatocytes (hPSC-Heps) remains a challenge. We hypothesized that the cellular microenvironment plays a vital role in the maturation of immature hepatocytes. In this study, we examined the role of mechanical stiffness, a key component of the cellular microenvironment, in the maturation of hPSC-Heps. We cultured hPSC-Heps on collagen-coated polyacrylamide hydrogels with varying elastic moduli. On softer substrates the hPSC-Heps formed compact colonies while on stiffer substrates they formed a diffuse monolayer. We observed an inverse correlation between albumin production and substrate stiffness. The expression of key cytochrome enzymes, which are expressed at higher levels in the adult liver compared to the fetal liver, also correlated inversely with substrate stiffness, whereas fetal markers such as Cyp3A7 and AFP showed no correlation with stiffness. Culture of hPSC-Heps on soft substrates for 12 days led to 10−30 fold increases in the expression of drug-metabolizing enzymes. These results demonstrate that substrate stiffness similar to that of the liver enables aspects of the maturation of hPSC-Heps.

152. Koh, W.L., Tham, P.H., Yu, H., Leo, H.L., and Kah, J.C.Y. (2016) Aggregation and Protein Corona on Gold Nanoparticles Affect Viability and Liver Functions of Primary Rat Hepatocytes. Nanomedicine, September 2016; 11(17): 2275-2287. DOI: 10.2217/nnm-2016-0173

We examined the impact of aggregation and protein corona formation of gold nanoparticles (AuNPs) on the cytotoxicity, uptake and metabolism, specifically urea and albumin synthesis, of primary rat hepatocytes. Materials & methods: The AuNPs were synthesized via citrate reduction and the human serum protein corona was preformed on the AuNPs. Primary hepatocytes were isolated from male Wistar rats via two-step in situ collagenase perfusion method, and were dosed with both citrate-capped (AuNP-Cit) and protein corona coated AuNPs (AuNP-Cor). Results: The AuNP-Cor showed higher cell uptake and reduced cell viability compared with aggregated AuNP-Cit. Urea and albumin secretions showed AuNP dose dependency. Both AuNP-Cit and AuNP-Cor exerted only an acute effect on the albumin synthesis of hepatocytes with no chronic impact.

151. Ng, I.C., Pawijit, P., Teo, L.Y., Li, H.P., Lee, S.Y., and Yu, H. (2016) Kinectin-dependent endoplasmic reticulum transport supports focal complex maturation for chemotaxis in shallow gradients. Journal of Cell Science, 129(13): 2660-2672. DOI: 10.1242/jcs.181768

Chemotaxis in shallow gradients is accomplished by preferential maintenance of protrusions oriented towards the chemoattractant; however, the mechanism of preferential maintenance is not known. Here, we test the hypothesis that kinectin-dependent endoplasmic reticulum (ER) transport supports focal complex maturation to preferentially maintain correctly oriented protrusions. We knocked down kinectin expression of MDA-MB-231 cells using small interfering RNA and observed that kinectin contributes to the directional bias but not the speed of cell migration. Kymograph analysis revealed that the extension of protrusions oriented towards the chemoattractant was not affected by kinectin knockdown, but maintenance was. Immunofluorescence staining and live cell imaging demonstrated that kinectin transports ER preferentially to protrusions oriented towards the chemoattractant. ER then promotes the maturation of focal complexes into focal adhesions to maintain these protrusions for chemotaxis. Our results show that kinectin-dependent ER distribution can be localized by chemoattractants and provide a mechanism for biased protrusion choices during chemotaxis in shallow gradients.

150. Tasnim, F., Toh, Y.-C., Qu, Y., Li, H., Phan, D., Narmada, B.C., Anantharayanan, A., Mittal, N., Meng, R.Q., and Yu, H. (2016) Functionally enhanced human stem cell derived hepatocytes in galactosylated cellulosic sponges for hepatotoxicity testing. Molecular Pharmaceutics, 8 May 2016; 13(6): 1947-1957. DOI: 10.1021/acs.molpharmaceut.6b00119

Pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) are an attractive alternative to primary human hepatocytes (PHHs) used in applications ranging from therapeutics to drug safety testing studies. It would be critical to improve and maintain mature hepatocyte functions of the hPSC-HLCs, especially for long-term studies. If 3D culture systems were to be used for such purposes, it would be important that the system can support formation and maintenance of optimal-sized spheroids for long periods of time, and can also be directly deployed in liver drug testing assays. We report the use of 3-dimensional (3D) cellulosic scaffold system for the culture of hPSC-HLCs. The scaffold has a macroporous network which helps to control the formation and maintenance of the spheroids for weeks. Our results show that culturing hPSC-HLCs in 3D cellulosic scaffolds increases functionality, as demonstrated by improved urea production and hepatic marker expression. In addition, hPSC-HLCs in the scaffolds exhibit a more mature phenotype, as shown by enhanced cytochrome P450 activity and induction. This enables the system to show a higher sensitivity to hepatotoxicants and a higher degree of similarity to PHHs when compared to conventional 2D systems. These results suggest that 3D cellulosic scaffolds are ideal for the long-term cultures needed to mature hPSC-HLCs. The mature hPSC-HLCs with improved cellular function can be continually maintained in the scaffolds and directly used for hepatotoxicity assays, making this system highly attractive for drug testing applications.

149. Li, H., Yang, H., Xue, X., Liu, X., Tian, F., Poh, Y., Cai, H., Lee, Y.H., Yu, H., Ong, S.P., and Cai, B. (2016) A Metabolomics Approach to Study the Dual Modulation by Characterization of Chemical Alteration during Processing of Gardeniae Fructus using UPLC-ESI-QTOF. Analytical Methods, 7 May 2016; 8(17): 3629-3635. DOI: 10.1039/c5ay03265b

Dual modulation is an interesting phenomenon that may occur during Chinese Materia Medica (CMM) processing whereby the crude and processed products have completely opposite therapeutic effects in vivo due to chemical component alteration. Therefore, a comprehensive study of the chemical alteration in order to shed light on the reason behind dual modulation is of critical importance. Metabolomics employs an untargeted approach to obtain an overview on secondary metabolites in multi-component systems using high resolution LC-MS2, which fulfills the requirement of a comprehensive analysis to clarify the mechanism of dual modulation. Gardeniae Fructus (GF), is one of the many widely used medicines across Asia, and its processed product exhibits completely opposite therapeutic effects on blood stasis. Therefore, we chose crude and processed GF to examine changes in seondary using UPLC-ESI-QTOF. In the subsequent chemometric analysis, both principal component analysis(PCA, unsupervised feature extraction) and orthogonal partial least-squares analysis (OPLSA, supervised feature extraction) were used to find out the chemical changes during processing. Iridoid Glycosides – jaminoside B, genipine-1-b-gentiobioside, 6a-hydroxygeniposide, and geniposide – and other ingredients such as mannitol and crocin were found to have decreased three to four fold in processed GF compared to crude GF, whereas another iridoid glycoside with a carboxyl group, mussaenosidic acid, was found to increase two fold in the processed product. This rapid yet reliable screening method can be also applied to other CMM to characterize the chemical changes and further explain the reasons behind dual modulation.

148. Xu, S., Kang, C.H., Gou, X., Peng, Q., Yan, J., Zhuo, S., Cheng, C.L., He, Y., Kang, Y., Xia, W., So. P.T.C., Welsch, R., Rajapakse, J.C., and Yu, H. (2015) Quantification of liver fibrosis via second harmonic imaging of the Glisson's capsule from liver surface. Journal of Biophotonics, April 2016; 9(4): 351-363. DOI: 10.1002/jbio.201500001

Liver surface is covered by a collagenous layer called the Glisson’s capsule. The structure of the Glisson’s capsule is barely seen in the biopsy samples for histology assessment, thus the changes of the collagen network from the Glisson's capsule during the liver disease progression are not well studied. In this report, we investigated whether non-linear optical imaging of the Glisson’s capsule at liver surface would yield sufficient information to allow quantitative staging of liver fibrosis. In contrast to conventional tissue sections whereby tissues are cut perpendicular to the liver surface and interior information from the liver biopsy samples were used, we have established a capsule index based on significant parameters extracted from the second harmonic generation (SHG) microscopy images of capsule collagen from anterior surface of rat livers. Thioacetamide (TAA) induced liver fibrosis animal models was used in this study. The capsule index is capable of differentiating different fibrosis stages, with area under receiver operating characteristics curve (AUC) up to 0.91, making it possible to quantitatively stage liver fibrosis via liver surface imaging potentially with endomicroscopy.

147. Li, Q., Zhang, Y., Pluchon, P., Robens, J., Herr, K., Mercade, M., Thiery, J.P., Yu, H., and Viasnoff, V. (2016) Environmental scaffolding guides lumen elongation by inducing anisotropic intercellular mechanical tension. Nature Cell Biology, April 2016; 18:311-318. DOI: 10.1038/ncb3310

The de novo formation of secretory lumens plays an important role during organogenesis. It involves the establishment of a cellular apical pole1 and the elongation of luminal cavities2. The molecular parameters controlling cell polarization have been heavily scrutinized3, 4, 5. In particular, signalling from the extracellular matrix (ECM) proved essential to the proper localization of the apical pole by directed protein transport6. However, little is known about the regulation of the shape and the directional development of lumen into tubes. We demonstrate that the spatial scaffolding of cells by ECM can control tube shapes and can direct their elongation. We developed a minimal organ approach comprising of hepatocyte doublets cultured in artificial microniches to precisely control the spatial organization of cellular adhesions in three dimensions. This approach revealed a mechanism by which the spatial repartition of integrin-based adhesion can elicit an anisotropic intercellular mechanical stress guiding the osmotically driven elongation of lumens in the direction of minimal tension. This mechanical guidance accounts for the different morphologies of lumen in various microenvironmental conditions.

146. Tong, W.H., Fang, Y., Yan, J., Hong, X., Singh, N.H.,Wang, S.R., Nugraha, B., Xia, L., Fong, E.L.S., Iliescu, C., and Yu, H. (2016) Constrained spheroids for prolonged hepatocyte culture, Biomaterials, February 2016; 80: 106-120.. DOI: 10.1016/j.biomaterials.2015.11.036

Liver-specific functions in primary hepatocytes can be maintained over extended duration in vitro using spheroid culture. However, the undesired loss of cells over time is still a major unaddressed problem, which consequently generates large variations in downstream assays such as drug screening. In static culture, the turbulence generated by medium change can cause spheroids to detach from the culture substrate. Under perfusion, the momentum generated by Stokes force similarly results in spheroid detachment. To overcome this problem, we developed a Constrained Spheroids (CS) culture system that immobilizes spheroids between a glass coverslip and an ultra-thin porous Parylene C membrane, both surface-modified with poly(ethylene glycol) and galactose ligands for optimum spheroid formation and maintenance. In this configuration, cell loss was minimized even when perfusion was introduced. When compared to the standard collagen sandwich model, hepatocytes cultured as CS under perfusion exhibited significantly enhanced hepatocyte functions such as urea secretion, and CYP1A1 and CYP3A2 metabolic activity. We propose the use of the CS culture as an improved culture platform to current hepatocyte spheroid-based culture systems.

145. Xia, L., Hong, X., Sakban, R.B., Qu, Y., Singh, N.H., McMillian, M., Dallas, S., Silva, J., Sensenhauser, C., Zhao, S., Lim, H.K., and Yu, H. (2016) Cytochrome P450 induction response in tethered spheroids as a three-dimensional human hepatocyte in vitro model. Journal of Applied Toxicology, February 2016; 36(2): 320-329.. DOI: 10.1002/jat.3189

Cytochrome P450 (CYP) induction is a key risk factor of clinical drug–drug interactions that has to bemitigated in the early phases of drug discovery. Three-dimensional (3D) cultures of hepatocytes in vitro have recently emerged as a potentially better platform to recapitulate the in vivo liver structure and to maintain long-term hepatic functions as compared with conventional two-dimensional (2D) monolayer cultures. However, the majority of published studies on 3D hepatocyte models use rat hepatocytes and the response to CYP inducers between rodents and humans is distinct. In the present study,we constructed tethered spheroids on RGD/galactose-conjugated membranes as an in vitro 3D model using cryopreserved human hepatocytes. CYP3A4mRNA expression in the tethered spheroidswas induced to a significantly greater extent than those in the collagen sandwich cultures, indicating the transcriptional regulation was more sensitive to the CYP inducers in the 3D model. Induction of CYP1A2, CYP2B6 and CYP3A4 activities in the tethered spheroids were comparable to, if not higher than that observed in the collagen sandwich cultures. The membrane-based model is readily integrated into multi-well plates for higher-throughput drug testing applications, which might be an alternative model to screen the CYP induction potential in vitro with more physiological relevance.

2015

144. Tan, W.J., Yan, J., Xu, S., Thike, A.A., Bay, B.H., Yu, H., Tan, M.-H., and Tan, P.H. (2015) Second harmonic generation microcopy is a novel technique for differential diagnosis of breast fibroepithelial lesions Journal of Clinical Pathology, December 2015; 68(12): 1033-1035.. DOI: 10.1136/jclinpath-2015-203231

Breast fibroepithelial lesions, including fibroadenomas and phyllodes tumours, are commonly encountered in clinical practice. As histological differences between these two related entities may be subtle, resulting in a challenging differential diagnosis, pathological techniques to assist the differential diagnosis of these two entities are of high interest. An accurate diagnosis at biopsy is important given corresponding implications for clinical decision-making including surgical extent and monitoring. Second harmonic generation (SHG) microscopy is a recently developed optical imaging technique capable of robust, powerful and unbiased label-free direct detection of collagen fibril structure in tissue without the use of antibodies. We constructed tissue microarrays emulating limited materials on biopsy to investigate quantitative collagen signal in fibroepithelial lesions using SHG microscopy. Archived formalin-fixed paraffin-embedded materials of 47 fibroepithelial lesions (14 fibroadenomas and 33 phyllodes tumours) were evaluated. Higher collagen signal on SHG microscopy was observed in fibroadenomas than phyllodes tumours on SHG imaging (p<0.001, area under the curve 0.859). At an automated threshold (2.5 million positive pixels), the sensitivity and specificity of the SHG microscopy for fibroadenoma classification was 71.4% and 84.4%, respectively. To corroborate these findings, we performed immunohistochemistry on tissue array sections using collagen I and III primary antibodies. Both collagen I and III immunohistochemical expressions were also significantly higher in fibroadenomas than in phyllodes tumours (p<0.001). In conclusion, label-free collagen quantitation on SHG microscopy is a novel imaging approach that can aid the differential diagnosis of fibroepithelial lesions.

143. Kathuria, H., Kochhar, J.S., Fong, M.H.M., Hashimoto, M., Iliescu, C., Yu, H., and Kang, L. (2015) Polymeric Microneedle Array Fabrication by Photolithography. Journal of Visualized Experiments, 5 Nov 2015; e52914. DOI: 10.3791/52914
Video available at http://www.jove.com/video/52914

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 μm ± 8.4 μm and 71.6 μm ± 13.7 μm, with two different lengths (1,336 μm ± 193 μm and 957 μm ± 171 μm) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

142. Qiu, L., Yu, H., and Liang, F. (2015) Multiple C2 domains transmembrane protein 1 is expressed in CNS neurons and possibly regulates cellular vesicle retrieval and oxidative stress. Journal of Neurochemistry, November 2015; 135(3): 492-507. DOI: 10.1111/jnc.13251

Multiple C2 domains transmembrane protein 1 (MCTP1) contains two transmembrane regions and three C2 domains of high Ca2+-binding affinity. Single-nucleotide polymorphism (SNP) of human MCTP1 gene is reportedly associated with bipolar disorder, but expression and function of MCTP1 in the CNS is still largely unknown. We cloned rat MCTP1 isoforms, and studied expression of MCTP1 transcript and protein in the CNS. Subcellular distribution and functional roles of MCTP1 were investigated in cultured primary neurons or PC12 cells by over-expression, cell imaging, and flow cytometry. MCTP1 immunostaining was seen in both CNS neuronal cell bodies and processes, especially in the hippocampus, dentate gyrus, medial habenular nucleus, amygdala, and selected cerebral and cerebellar cortical areas/layers. Under an electron microscope, MCTP1 immunoreactivity was observed on vesicles in neuronal cell bodies and pre-synaptic axon terminals. In cultured primary neurons and PC12 cells MCTP1 was detected on selected populations of secretory vesicles and endosomes. MCTP1 over-expression significantly inhibited neuronal transferrin endocytosis, secretory vesicle retrieval, cell migration, and oxidative stress from glutamate toxicity. Thus MCTP1 might be involved in regulating endocytic recycling of specific CNS neurons and synapses. MCTP1 abnormality might cause altered synaptic vesicle recycling, and thereby lead to vulnerability to neuropsychiatric diseases.

141. Raja, A.M., Xu, S., Zhuo, S., Tai, D.C.S., Sun, W., So, P.T.C., Welsh, R.E., Chen, C.-S., and Yu, H. (2015) Differential Remodeling of Extracellular Matrices by Breast Cancer Initiating Cells. Journal of Biophotonics, October 2015; 8(10): 804-815. DOI: 10.1002/jbio.201400079

Cancer initiating cells (CICs) have been the focus of recent anti-cancer therapies, exhibiting strong invasion capability via potentially enhanced ability to remodel extracellular matrices (ECM). We have identified CICs in a human breast cancer cell line, MX-1, and developed a xenograft model in SCID mice. We investigated the CICs’ matrix-remodeling effects using Second Harmonic Generation SHG) microscopy to identify potential phenotypic signatures of the CIC-rich tumors. The isolated CICs exhibit higher proliferation, drug efflux and drug resistant properties in vitro; were more tumorigenic than non-CICs, resulting in more and larger tumors in the xenograft model. The CIC-rich tumors have less collagen in the tumor interior than in the CIC-poor tumors supporting the idea that the CICs can remodel the collagen more effectively. The collagen fibers were preferentially aligned perpendicular to the CIC-rich tumor boundary while parallel to the CIC-poor tumor boundary suggesting more invasive behavior of the CIC-rich tumors. These findings would provide potential translational values in quantifying and monitoring CIC-rich tumors in future anti-cancer therapies.

140. Tasnim, F., Phan, D., Toh, Y.-C., and Yu, H. (2015) Cost-effective differentiation of hepatocyte-like cells from human pluripotent stem cells using small molecules. Biomaterials, November 2015; 70: 115-125. DOI: 10.1016/j.biomaterials.2015.08.002

Significant efforts have been invested into the differentiation of stem cells into functional hepatocyte-like cells that can be used for cell therapy, disease modeling and drug screening. Most of these efforts have been concentrated on the use of growth factors to recapitulate developmental signals under in vitro conditions. Using small molecules instead of growth factors would provide an attractive alternative since small molecules are cell-permeable and cheaper than growth factors. We have developed a protocol for the differentiation of human embryonic stem cells into hepatocyte-like cells using a predominantly small moleculeebased approach (SM-Hep). This 3 step differentiation strategy involves the use of optimized concentrations of LY294002 and bromo-indirubin-3’-oxime (BIO) for the generation of definitive endoderm; sodium butyrate and dimethyl sulfoxide (DMSO) for the generation of hepatoblasts and SB431542 for differentiation into hepatocyte-like cells. Activin A is the only growth factor required in this protocol. Our results showed that SM-Hep were morphologically and functionally similar or better compared to the hepatocytes derived from the growth-factor induced differentiation (GF-Hep) in terms of expression of hepatic markers, urea and albumin production and cytochrome P450 (CYP1A2 and CYP3A4) activities. Cell viability assays following treatment with paradigm hepatotoxicants Acetaminophen, Chlorpromazine, Diclofenac, Digoxin, Quinidine and Troglitazone showed that their sensitivity to these drugs was similar to human primary hepatocytes (PHHs). Using SM-Hep would result in 67% and 81% cost reduction compared to GF-Hep and PHHs respectively. Therefore, SM-Hep can serve as a robust and cost effective replacement for PHHs for drug screening and development.

139. Iliescu, C., Xu, G., Tong, W.H., Fang, Y., Balan, C.M., Tresset, G., and Yu, H. (2015) Cell patterning using a dielectrophoretic-hydrodynamic trap. Microfluidics and Nanofluidics, August 2015; 19(2): 363-373.. DOI: 10.1007/s10404-015-1568-2

The paper presents a dielectrophoretic method for cell patterning using dielectrophoretic–hydrodynamic trap. A distinctive characteristic of the device is that the dielectrophoretic (DEP) force is generated using a structure that combines conventional electrode-based DEP (eDEP) with insulator-based DEP method (iDEP). The conventional eDEP force is generated across the microfluidic channel between a top plate indium tin oxide electrode and a thin CrAu electrode. Meantime, an isolating cage built from SU8 photoresist around the thin electrode modifies the electric field generating an iDEP force. The cells that are flowing through a microfluidic channel are trapped in the SU8 cage by the total DEP force. As a result, according to the cell dimension and the thickness of the SU8 layer, different cell patterns can be achieved. If the cell’s size is sensitively smaller than the dimensions of the hydrodynamic trap, due to the dipole–dipole interaction, the cell can be organized in 3D structures. The trapping method can be used for conducting genetic, biochemical or physiological studies on cells.

138. Wang, Z., Luo, X., Anene-Nzelu, C., Yu, Y., Hong, X., Singh, N.H., Xia, L., Liu, S., and Yu, H. (2015) HepaRG Culture in Tethered Spheroids as an in vitro Three-dimensional Model for Drug Safety Screening. Journal of Applied Toxicology, August 2015: 35(7): 909-917. DOI: 10.1002/jat.3090

Conventional two-dimensional (2D) monolayer cultures of HepaRG cells allow in vitro maintenance of many liver-specific functions. However, cellular dedifferentiation and functional deterioration over an extended culture period in the conventional 2D HepaRG culture have hampered its applications in drug testing. To address this issue, we developed tethered spheroids of HepaRG cells on Arg–Gly–Asp (RGD) and galactose-conjugated substratum with an optimized hybrid ratio as an in vitro three-dimensional (3D) human hepatocyte model. The liver-specific gene expression level and drug metabolizing enzyme activities in HepaRG-tethered spheorids were markedly higher than those in 2D cultures throughout the culture period of 7 days. The inducibility of three major cytochrome P450 (CYP) enzymes, namely CYP1A2, CYP2B6 and CYP3A4, was improved in both mRNA and activity level in tethered spheroids. Drug-induced cytotoxic responses to model hepatotoxins (acetaminophen, chlorpromazine and ketoconazole) in tethered spheroids were comparable to 2D cultures as well as other studies in the literature. Our results suggested that the HepaRG-tethered spheroid would be an alternative in vitro model suitable for drug safety screening.

137. Xing, J., Toh, Y.-C., Xu, S., and Yu, H. (2015) A method for human teratogen detection by geometrically confined cell differentiation and migration. Scientific Reports, 12 May 2015; 5: 10038. DOI: 10.1038/srep10038

Unintended exposure to teratogenic compounds can lead to various birth defects; however current animal-based testing is limited by time, cost and high inter-species variability. Here, we developed a human-relevant in vitro model, which recapitulated two cellular events characteristic of embryogenesis, to identify potentially teratogenic compounds. We spatially directed mesoendoderm differentiation, epithelial-mesenchymal transition and the ensuing cell migration in micropatterned human pluripotent stem cell (hPSC) colonies to collectively form an annular mesoendoderm pattern. Teratogens could disrupt the two cellular processes to alter the morphology of the mesoendoderm pattern. Image processing and statistical algorithms were developed to quantify and classify the compounds’ teratogenic potential. We not only could measure dose-dependent effects but also correctly classify species-specific drug (Thalidomide) and false negative drug (D-penicillamine) in the conventional mouse embryonic stem cell test. This model offers a scalable screening platform to mitigate the risks of teratogen exposures in human.

136. Toh, Y.C., Xing, J., and Yu, H. (2015) Modulation of integrin and E-cadherin-mediated adhesions to spatially control heterogeneity in human pluripotent stem cell differentiation. Biomaterials, May 2015; 50: 87-97. DOI: 10.1016/j.biomaterials.2015.01.019

Heterogeneity in human pluripotent stem cell (PSC) fates is partially caused by mechanical asymmetry arising from spatial polarization of cell–cell and cell-matrix adhesions. Independent studies have shown that integrin and E-cadherin adhesions promote opposing differentiation and pluripotent fates respectively although their crosstalk mechanism in modulating cell fate heterogeneity remains unknown. Here, we demonstrated that spatial polarization of integrin and E-cadherin adhesions in a human PSC colony compete to recruit Rho-ROCK activated myosin II to different localities to pattern pluripotent-differentiation decisions, resulting in spatially heterogeneous colonies. Cell micropatterning was used to modulate the spatial polarization of cell adhesions, which enabled us to prospectively determine localization patterns of activated myosin II and mesoendoderm differentiation. Direct inhibition of Rho-ROCK-myosin II activation phenocopied E-cadherin rather than integrin inhibition to form uniformly differentiated colonies. This indicated that E-cadherin was the primary gatekeeper to differentiation progression. This insight allows for biomaterials to be tailored for human PSC maintenance or differentiation with minimal heterogeneity.

 

135. Peng, Q., Zhuo, S., So, P., and Yu, H. (2015) Improving liver fibrosis diagnosis based on forward and backward second harmonic generation signals. Applied Physics Letters, 26 February 2015; 106(8): 083701. DOI: 10.1063/1.4913907

The correlation of forward second harmonic generation (SHG) signal and backward SHG signal in different liver fibrosis stages was investigated. We found that three features, including the collagen percentage for forward SHG, the collagen percentage for backward SHG, and the average intensity ratio of two kinds of SHG signals, can quantitatively stage liver fibrosis in thioacetamide-induced rat model. We demonstrated that the combination of all three features by using a support vector machine classification algorithm can provide a more accurate prediction than each feature alone in fibrosis diagnosis.

 

 

 

 

2014

134. Yan, J., Kang, Y., Xu, S., Ong, S.L.L., Zhuo, S., Bunte, R.M., Chen, N., Asada, H.H., So, P., Wanless, I.R., and Yu, H. (2014) In vivo label-free quantification of liver microcirculation using dual modality microscopy. Journal of Biomedical Optics, 11 November 2014; 19(11): 116006. DOI: 10.1117/1.JBO.19.11.116006

Microcirculation lesion is a common symptom of chronic liver diseases in the form of vasculature deformation and circulation alteration. In acute to chronic liver diseases such as biliary atresia, microcirculation lesion can have an early onset. Detection of microcirculation lesion is meaningful for studying the progression of liver disease. We have combined wide-field fluorescence microscopy and a laser speckle contrast technique to characterize hepatic microcirculation in vivo without labeling in a bile-duct ligation rat fibrosis model of biliary atresia. Through quantitative image analysis of four microcirculation parameters, we observed significant microcirculation lesion in the early to middle stages of fibrosis. This bimodal imaging method is useful to assess hepatic microcirculation lesion for the study of liver diseases.

133. Iliescu, C., Marculescu, C., Venkataraman, S., Languille, B., Yu, H., and Tresset, G. (2014) On-Chip Controlled Surfactant-DNA Coil-Globule Transition by Rapid Solvent Exchange Using Hydrodynamic Flow Focusing. Langmuir, 29 October 2014; 30(44): 13125-13136. DOI: 10.1021/la5035382

This paper presents a microfluidic method for precise control of the size and polydispersity of surfactant−DNA nanoparticles. A mixture of surfactant and DNA dispersed in 35% ethanol is focused between two streams of pure water in a microfluidic channel. As a result, a rapid change of solvent quality takes place in the central stream, and the surfactant-bound DNA molecules undergo a fast coil−globule transition. By adjusting the concentrations of DNA and surfactant, fine-tuning of the nanoparticle size, down to a hydrodynamic diameter of 70 nm with a polydispersity index below 0.2, can be achieved with a good reproducibility.

132. Yin, L., Zheng, D., Limmon, G.V., Leung, N.H.N., Xu, S., Rajapakse, J.C., Yu, H., Chow V.T.K., and Chen, J. (2014) Aging Exacerbates Damage and Delays Repair of Alveolar Epithelia following Influenza Viral Pneumonia. Respiratory Research, 30 September 2014; 15(1):116. DOI: 10.1186/s12931-014-0116-z

Background: Influenza virus infection causes significantly higher levels of morbidity and mortality in the elderly. Studies have shown that impaired immunity in the elderly contributes to the increased susceptibility to influenza virus infection, however, how aging affects the lung tissue damage and repair has not been completely elucidated. Methods: Aged (16–18 months old) and young (2–3 months old) mice were infected with influenza virus intratracheally. Body weight and mortality were monitored. Different days after infection, lung sections were stained to estimate the overall lung tissue damage and for club cells, pro-SPC+ bronchiolar epithelial cells, alveolar type I and II cells to quantify their frequencies using automated image analysis algorithms. Results: Following influenza infection, aged mice lose more weight and die from otherwise sub-lethal influenza infection in young mice. Although there is no difference in damage and regeneration of club cells between the young and the aged mice, damage to alveolar type I and II cells (AT1s and AT2s) is exacerbated, and regeneration of AT2s and their precursors (pro-SPC-positive bronchiolar epithelial cells) is significantly delayed in the aged mice. We further show that oseltamivir treatment reduces virus load and lung damage, and promotes pulmonary recovery from infection in the aged mice. Conclusions: These findings show that aging increases susceptibility of the distal lung epithelium to influenza infection and delays the emergence of pro-SPC positive progenitor cells during the repair process. Our findings also shed light on possible approaches to enhance the clinical management of severe influenza pneumonia in the elderly.

131. Venkatesh, S.K., Xu, S., Tai, D., Yu, H., and Wee, A. (2014) Correlation of MR elastography with morphometric quantification of liver fibrosis (Fibro-C-Index) in chronic hepatitis B. Magnetic Resonance in Medicine, October 2014; 72(4): 1123-1129. DOI: 10.1002/mrm.25002

PURPOSE: We evaluated the correlation of MR Elastography (MRE) with morphometric assessment of liver fibrosis in chronic hepatitis B (CHB). METHODS: Thirty-two patients with CHB underwent both MRE and a liver biopsy within a 6-month interval. MRE was performed using standard MRE sequence on a 1.5 Tesla clinical scanner. The liver stiffness (LS) was measured on automatically generated stiffness maps. Morphometric quantification of fibrosis of liver biopsies was performed using a semi-automated image analysis program and expressed as percentage area (Fibro-C-Index). Correlations between MRE, Fibro-C-Index, and histologic fibrosis stages were evaluated. Receiver operating curve (ROC) analysis of MRE and Fibro-C-index for differentiating fibrosis (≥F1), significant fibrosis (≥F2), advanced fibrosis (≥F3), and cirrhosis (F4) was performed. RESULTS: MRE showed excellent correlation with both Fibro-C-Index (r=0.78, 95% confidence interval [CI] 0.59-0.88, P<0.001) and histologic staging (rho=0.87, 95% CI, 0.72-0.94, P<0.0001). Significant differences in MRE (P=0.0001) and Fibro-C-Index (P=0.003) among different stages of liver fibrosis was found. MRE and Fibro-C-Index had similar accuracies for differentiating fibrosis stages: ≥F1 (0.87 versus 0.81, P=0.6), ≥F2 (0.95 versus 0.94, P=0.78), ≥F3 (0.98 versus 0.96, P=0.76), and F4 (1.00 versus 0.92, P=0.10). CONCLUSION: MRE is an excellent noninvasive indicator of liver fibrosis burden in CHB.

130. Kizhmuri, D.P., Sreejith, S., Pichandi, A., Kang, Y., Peng, Q., Maji, S.K., Tong, Y., Yu, H., Zhao, Y., Ramamurthy, P., and Ajayaghosh, A. (2014) A Ratiometric Fluorescent Molecular Probe with Enhanced Two-photon Response upon Zn2+ Binding for in vitro and in vivo Bioimaging. Chemical Science, 1 September 2014; 5(9): 3469-3474. DOI: 10.1039/C4SC00736K

A bipyridine centered donor–acceptor–donor (D–p–A–p–D) type ratiometric fluorescent molecular probe exhibited an unprecedented enhancement in the two-photon absorption (2PA) cross section upon Zn2+ binding. Moreover, owing to the excited state charge-transfer of the fluorophore p-backbone, a significant enhancement in the two-photon (2P) excited fluorescence intensity was observed upon Zn2+ binding, resulting in a 13-fold enhancement in the 2PA cross section and a 9-fold enhancement in fluorescence brightness at 620 nm when compared to the cation-free fluorophore. The large 2PA cross section of 1433 GM and 2P action cross section (860 GM), with an excellent 2P excited fluorescence variation from 517 to 620 nm upon Zn2+ binding, facilitated the ratiometric monitoring of free zinc ions in cells. The low cytotoxicity and good photostability of the fluorophore allowed two-photon Zn2+ imaging of HeLa cells. In addition, in vivo two-photon imaging of Zn2+ ions in hepatocytes of live rats illustrated the viability of the probe in tissue imaging and monitoring of free zinc ions in live cells.

129. Xu, S., Wang, Y., Tai. D.C.S., Wang, S., Cheng, C.L., Peng, Q., Yan, J., Chen, Y., Sun, J., Liang, X., Zhu, Y., Rajapakse, J.C., Welsch, R., So, P.T.C., Wee, A., Hou, J., and Yu, H. (2014) qFibrosis: A fully-quantitative innovative method incorporating histological features to facilitate accurate fibrosis scoring in animal model and chronic hepatitis B patients. Journal of Hepatology, August 2014; 61(2): 260-269. DOI: 10.1016/j.jhep.2014.02.015

Background & Aims: There is increasing need for accurate assessment of liver fibrosis/cirrhosis. We aimed to develop qFibrosis, a fully-automated assessment method combining quantification of histopathological architectural features, to address unmet needs in core biopsy evaluation of fibrosis in chronic hepatitis B (CHB) patients. Methods: qFibrosis was established as a combined index based on 87 parameters of architectural features. Images acquired from 25 Thioacetamide-treated rat samples and 162 CHB core biopsies were used to train and test qFibrosis and to demonstrate its reproducibility. qFibrosis scoring was analyzed employing Metavir and Ishak fibrosis staging as standard references, and collagen proportionate area (CPA) measurement for comparison. Results: qFibrosis faithfully and reliably recapitulates Metavir fibrosis scores, as it can identify differences between all stages in both animal samples (p <0.001) and human biopsies (p <0.05). It is robust to sampling size, allowing for discrimination of different stages in samples of different sizes (area under the curve (AUC): 0.93–0.99 for animal samples: 1–16 mm2; AUC: 0.84–0.97 for biopsies: 10–44 mm in length). qFibrosis can significantlypredict staging underestimation in suboptimal biopsies (<15 mm) and under- and over-scoring by different pathologists (p <0.001). qFibrosis can also differentiate between Ishak stages 5 and 6 (AUC: 0.73, p = 0.008), suggesting the possibility of monitoring intra-stage cirrhosis changes. Best of all, qFibrosis demonstrates superior performance to CPA on all counts. Conclusions: qFibrosis can improve fibrosis scoring accuracy and throughput, thus allowing for reproducible and reliable analysis of efficacies of anti-fibrotic therapies in clinical research and practice.

128. Zhuo, S., Yan, J., Kang, Y., Xu, S., Peng, Q., So, P.T.C., and Yu, H. (2014) In vivo, label-free, three-dimensional quantitative imaging of liver surface using multi-photon microscopy. Applied Physics Letters, 16 July 2014; 105(2): 023701. DOI: 110.1063/1.4890593

Various structural features on the liver surface reflect functional changes in the liver. The visualization of these surface features with molecular specificity is of particular relevance to understanding the physiology and diseases of the liver. Using multi-photon microscopy (MPM), we have developed a label-free, three-dimensional quantitative and sensitive method to visualize various structural features of liver surface in living rat. MPM could quantitatively image the microstructural features of liver surface with respect to the sinuosity of collagen fiber, the elastic fiber structure, the ratio between elastin and collagen, collagen content, and the metabolic state of the hepatocytes that are correlative with the pathophysiologically induced changes in the regions of interest. This study highlights the potential of this technique as a useful tool for pathophysiological studies and possible diagnosis of the liver diseases with further development.

127. Ananthanarayanan,A., Nugraha, B., Triyatni,M., Hart, S., Sankuratri, S., and Yu, H. (2014) Scalable Spheroid Model of Human Hepatocytes for Hepatitis C Infection and Replication. Molecular Pharmaceutics, 7 July 2014; 11(7): 2106-2114. DOI: 10.1021/mp500063y

Developing effective new drugs against hepatitis C (HCV) virus has been challenging due to the lack of appropriate small animal and in vitro models recapitulating the entire life cycle of the virus. Current in vitro models fail to recapitulate the complexity of human liver physiology. Here we present a method to study HCV infection and replication on spheroid cultures of Huh 7.5 cells and primary human hepatocytes. Spheroid cultures are constructed using a galactosylated cellulosic sponge with homogeneous macroporosity, enabling the formation and maintenance of uniformly sized spheroids. This facilitates easy handling of the tissue-engineered constructs and overcomes limitations inherent of traditional spheroid cultures. Spheroids formed in the galactosylated cellulosic sponge show enhanced hepatic functions in Huh 7.5 cells and maintain liver-specific functions of primary human hepatocytes for 2 weeks in culture. Establishment of apical and basolateral polarity along with the expression and localization of all HCV specific entry proteins allow for a 9-fold increase in viral entry in spheroid cultures over conventional monolayer cultures. Huh 7.5 cells cultured in the galactosylated cellulosic sponge also support replication of the HCV clone, JFH (Japanese fulminant hepatitis)-1 at higher levels than in monolayer cultures. The advantages of our system in maintaining liver-specific functions and allowing HCV infection together with its ease of handling make it suitable for the study of HCV biology in basic research and pharmaceutical R&D.

126. Cha, J.W., Singh, V.R., Kim, K.H., Subrmanian, J., Peng, Q., Yu, H., Nedivi, E., and So, P.T.C. (2014) Reassignment of Scattered Emission Photons in Multifocal Multiphoton Microscopy. Scientific Reports, 5 June 2014; 4: 5153. DOI: 10.1038/srep05153

Multifocal multiphoton microscopy (MMM) achieves fast imaging by simultaneously scanning multiple foci across different regions of specimen. The use of imaging detectors in MMM, such as CCD or CMOS, results in degradation of image signal-to-noise-ratio (SNR) due to the scattering of emitted photons. SNR can be partly recovered using multianode photomultiplier tubes (MAPMT). In this design, however, emission photons scattered to neighbor anodes are encoded by the foci scan location resulting in ghost images. The crosstalk between different anodes is currently measured a priori, which is cumbersome as it depends specimen properties. Here, we present the photon reassignment method for MMM, established based on the maximum likelihood (ML) estimation, for quantification of crosstalk between the anodes of MAPMTwithout a priori measurement. The method provides the reassignment of the photons generated by the ghost images to the original spatial location thus increases the SNR of the final reconstructed image.

125. Wang, J., Tucker-Kellogg, L., Ng, I.C., Jia, R., Thiagarajan, P.S., White, J.K., and Yu, H. (2014) The Self-Limiting Dynamics of TGF-β Signaling In Silico and In Vitro, with Negative Feedback through PPM1A Upregulation. PLoS Computational Biology, 5 June 2014; 10(6): e1003573. DOI: 10.1371/journal.pcbi.1003573

The TGF-b/Smad signaling system decreases its activity through strong negative regulation. Several molecular mechanisms of negative regulation have been published, but the relative impact of each mechanism on the overall system is unknown. In this work, we used computational and experimental methods to assess multiple negative regulatory effects on Smad signaling in HaCaT cells. Previously reported negative regulatory effects were classified by time-scale: degradation of phosphorylated R-Smad and I-Smad-induced receptor degradation were slow-mode effects, and dephosphorylation of RSmad was a fast-mode effect. We modeled combinations of these effects, but found no combination capable of explaining the observed dynamics of TGF-b/Smad signaling. We then proposed a negative feedback loop with upregulation of the phosphatase PPM1A. The resulting model was able to explain the dynamics of Smad signaling, under both short and long exposures to TGF-b. Consistent with this model, immuno-blots showed PPM1A levels to be significantly increased within 30 min after TGF-b stimulation. Lastly, our model was able to resolve an apparent contradiction in the published literature, concerning the dynamics of phosphorylated R-Smad degradation. We conclude that the dynamics of Smad negative regulation cannot be explained by the negative regulatory effects that had previously been modeled, and we provide evidence for a new negative feedback loop through PPM1A upregulation. This work shows that tight coupling of computational and experiments approaches can yield improved understanding of complex pathways.

124. Stanciu, S.G., Xu, S., Peng, Q., Yan, J., Stanciu, G.A., Welsch, R.E., So, P.T.C., Csucs, G., and Yu, H. (2014) Experimenting Liver Fibrosis Diagnostic by Two Photon Excitation Microscopy and Bag-of-Features Image Classification. Scientific Reports, 10 April 2014; 4: 4636. DOI: 10.1038/srep04636

The accurate staging of liver fibrosis is of paramount importance to determine the state of disease progression, therapy responses, and to optimize disease treatment strategies. Non-linear optical microscopy techniques such as two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) can image the endogenous signals of tissue structures and can be used for fibrosis assessment on non-stained tissue samples. While image analysis of collagen in SHG images was consistently addressed until now, cellular and tissue information included in TPEF images, such as inflammatory and hepatic cell damage, equally important as collagen deposition imaged by SHG, remain poorly exploited to date. We address this situation by experimenting liver fibrosis quantification and scoring using a combined approach based on TPEF liver surface imaging on a Thioacetamide-induced rat model and a gradient based Bag-of-Features (BoF) image classification strategy. We report the assessed performance results and discuss the influence of specific BoF parameters to the performance of the fibrosis scoring framework.

123. Poenar, D.P., Iliescu, C., Boulaire, J., and Yu, H. (2014) Label-free virus identification and characterization using electrochemical impedance spectroscopy. Electrophoresis, February 2014; 35(2-3): 433-440. DOI: 10.1002/elps.201300368

We demonstrate here the application of electrochemical impedance spectroscopy (EIS) in microfluidic devices for label-free virus identification by means of their specific “signature” and also investigate its feasibility for titer quantitation using two basic approaches. The first one is a method based on identifying so-called “resonance” frequencies manifesting in our microdevices and monitoring their variation as a function of the virus concentration, whereas the second one relies on measuring the relative impedance variation at these “resonance” frequencies. Best results have been obtained for the highest “resonance” frequency (∼80 MHz), which we attribute to be due to both the structure of the microdevice and the extremely small size of the viruses that make their effect significant only at such frequencies. This is a simpler method of determining virus concentration in diluted solutions of purified viruses than the well-established traditional plaque assay titer estimation method, and—since it is based on frequency measurement—could potentially be more accurate.

2013

122. Anene-Nzelu, C.G., Peh, K.Y., Fraiszudeen, A., Kuan, Y.H., Ng, S.H., Toh, Y.C., Leo, H.L., and Yu, H. (2013) Scalable alignment of three-dimensional cellular constructs in a microfluidic chip. Lab on a Chip, 23 Aug 2013; 13(20): 4124-4133. DOI: 10.1039/c3lc50730k

There have been considerable efforts to engineer three-dimensional (3D) microfluidic environments to enhance cellular function over conventional two-dimensional (2D) cultures in microfluidic chips, but few involve topographical features, such as micro/nano-grooves, which are beneficial for cell types of cardiac, skeletal and neuronal lineages. Here we have developed a cost-effective and scalable method to incorporate micro-topographical cues into microfluidic chips to induce cell alignment. Using commercially available optical media as molds for replica molding, we produced large surface areas of polydimethylsiloxane (PDMS) micro-grooved substrates and plasma-bonded them to multiple microfluidic chips. Besides aligning a 2D monolayer of cells, the micro-grooved substrate can align 3D cellular constructs on chip. C2C12 mouse myoblasts were cultured three-dimensionally in a microfluidic chip with incorporated PDMS micro-grooved substrate remodeled into an aligned 3D cellular construct, where the actin cytoskeleton and nuclei were preferentially oriented along the micro-grooves. Cells within the 3D cellular constructs can align without being in direct contact with the micro-grooves due to synergism between topography and fluid shear stress. Aligned C2C12 3D cellular constructs showed enhanced differentiation into skeletal muscles as compared to randomly aligned ones. This novel method enables the routine inclusion of micro-topographical cues into 2D or 3D microfluidic cultures to generate relevant physiological models for studying tissue morphogenesis and drug screening applications.

121. Zheng, D.H., Limmon, G.V., Yin, L., Leung, N.H.N., Yu, H., Chow. V.T.K., and Chen, J.Z. (2013) A Cellular Pathway Involved in Clara Cell to Alveolar Type II Cell Differentiation after Severe Lung Injury. PLoS ONE, 5 Aug 2013; 8(8): e71028. DOI: 10.1371/journal.pone.0071028

Regeneration of alveolar epithelia following severe pulmonary damage is critical for lung function. We and others have previously shown that Scgb1a1-expressing cells, most likely Clara cells, can give rise to newly generated alveolar type 2 cells (AT2s) in response to severe lung damage induced by either influenza virus infection or bleomycin treatment. In this study, we have investigated cellular pathway underlying the Clara cell to AT2 differentiation. We show that the initial intermediates are bronchiolar epithelial cells that exhibit Clara cell morphology and express Clara cell marker, Scgb1a1, as well as the AT2 cell marker, pro-surfactant protein C (pro-SPC). These cells, referred to as pro-SPC+ bronchiolar epithelial cells (or SBECs), gradually lose Scgb1a1 expression and give rise to pro-SPC+ cells in the ring structures in the damaged parenchyma, which appear to differentiate into AT2s via a process sharing some features with that observed during alveolar epithelial development in the embryonic lung. These findings suggest that SBECs are intermediates of Clara cell to AT2 differentiation during the repair of alveolar epithelia following severe pulmonary injury.

120. Saldutti, L.P., Beyer, B.K., Breslin, W., Brown, T.R., Chapin, R.E., Campion, S., Enright, B., Faustman, E., Foster, P.M.D., Hartung, T., Kelce, W., Kim, J.H., Loboa, E.G., Piersma, A.H., Seyler, D., Turner, K.J., Yu, H., Yu, X., and Sasaki, J.C. (2013) In vitro Testicular Toxicity Models: Opportunities for Advancement via Biomedical Engineering Techniques. ALTEX Alternatives to Animal Experimentation, 30(3): 353-377. DOI: 10.14573/altex.2013.3.353

To address the pressing need for better in vitro testicular toxicity models, a workshop sponsored by the International Life Sciences Institute (ILSI), the Health and Environmental Science Institute (HESI), and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT), was held at the Mt. Washington Conference Center in Baltimore, MD, USA on October 26-27, 2011. At this workshop, experts in testis physiology, toxicology, and tissue engineering discussed approaches for creating improved in vitro environments that would be more conducive to maintaining spermatogenesis and steroidogenesis and could provide more predictive models for testicular toxicity testing. This workshop report is intended to provide scientists with a broad overview of relevant testicular toxicity literature and to suggest opportunities where bioengineering principles and techniques could be used to build improved in vitro testicular models for safety evaluation. Tissue engineering techniques could, conceivably, be immediately implemented to improve existing models. However, it is likely that in vitro testis models that use single or multiple cell types will be needed to address such endpoints as accurate prediction of chemically induced testicular toxicity in humans, elucidation of mechanisms of toxicity, and identification of possible biomarkers of testicular toxicity.

119. Anene-Nzelu, C.G., Choudhury, D., Li, H., Fraiszudeen, A., Peh, K.Y., Toh, Y.-C., Ng, S.H., Leo, H.L., and Yu, H. (2013) Scalable cell alignment on optical media substrates. Biomaterials, July 2013; 34(21): 5078-5087. DOI: 10.1016/j.biomaterials.2013.03.070

Cell alignment by underlying topographical cues has been shown to affect important biological processes such as differentiation and functional maturation in vitro. However, the routine use of cell culture substrates with micro- or nano-topographies, such as grooves, is currently hampered by the high cost and specialized facilities required to produce these substrates. Here we present cost-effective commercially available optical media as substrates for aligning cells in culture. These optical media, including CD-R, DVD-R and optical grating, allow different cell types to attach and grow well on them. The physical dimension of the grooves in these optical media allowed cells to be aligned in confluent cell culture with maximal cellecell interaction and these cell alignment affect the morphology and differentiation of cardiac (H9C2), skeletal muscle (C2C12) and neuronal (PC12) cell lines. The optical media is amenable to various chemical modifications with fibronectin, laminin and gelatin for culturing different cell types. These low-cost commercially available optical media can serve as scalable substrates for research or drug safety screening applications in industry scales.

118. Narmada, B.C., Kang, Y., Venkatraman, L., Peng, Q., Sakban, R.B., Nugraha, B., Jiang, X., Bunte, R.M., So, P.T.C., Tucker-Kellogg, L., Mao, H.-Q., and Yu, H. (2013) Hepatic Stellate Cell–Targeted Delivery of Hepatocyte Growth Factor Transgene via Bile Duct Infusion Enhances Its Expression at Fibrotic Foci to Regress Dimethylnitrosamine-Induced Liver Fibrosis. Human Gene Therapy, 15 May 2013; 24(5): 508-519. DOI: 10.1089/hum.2012.158

Liver fibrosis generates fibrotic foci with abundant activated hepatic stellate cells and excessive collagen deposition juxtaposed with healthy regions. Targeted delivery of antifibrotic therapeutics to hepatic stellate cells (HSCs) might improve treatment outcomes and reduce adverse effects on healthy tissue. We delivered the hepatocyte growth factor (HGF) gene specifically to activated hepatic stellate cells in fibrotic liver using vitamin A–coupled liposomes by retrograde intrabiliary infusion to bypass capillarized hepatic sinusoids. The anti- fibrotic effects of DsRed2-HGF vector encapsulated within vitamin A–coupled liposomes were validated by decreases in fibrotic markers in vitro. Fibrotic cultures transfected with the targeted transgene showed a significant decrease in fibrotic markers such as transforming growth factor-b1. In rats, dimethylnitrosamineinduced liver fibrosis is manifested by an increase in collagen deposition and severe defenestration of sinusoidal endothelial cells. The HSC-targeted transgene, administered via retrograde intrabiliary infusion in fibrotic rats, successfully reduced liver fibrosis markers alpha-smooth muscle actin and collagen, accompanied by an increase in the expression of DsRed2-HGF near the fibrotic foci. Thus, targeted delivery of HGF gene to hepatic stellate cells increased the transgene expression at the fibrotic foci and strongly enhanced its antifibrotic effects.

117. Yin, L., Xu, S., Cheng, J., Zheng, D., Limmon, G. V., Leung, N.H.N., Rajapakse, J.C., Chow, V.T.K., Chen, J., and Yu, H. (2013) Spatiotemporal Quantification of Cell Dynamics in the Lung following Influenza Virus Infection. Journal of Biomedical Optics, 1 April 2013; 18(4): 046001. DOI: 10.1021/bc3005842

Although fibrous collagens are major structural components of extracellular matrix in mammals, collagen overproduction is associated with many human diseases including cancers and fibrosis. Collagen is typically identified in biomedical research by Western blot and immunohistochemistry; however, anticollagen antibodies employed in these analyses are difficult to prepare and their affinities to collagen can diminish if collagen becomes denatured during analyses. Previously, we discovered that single-stranded collagen mimetic peptides [CMPs, sequence: (GlyProHyp)9] can bind to denatured collagen chains by triple helix hybridization. Here, we present collagen-specific staining methods using simple CMPs conjugated to common fluorophores (e.g., carboxyfluorescein), which allow direct detection of collagens and collagen like proteins in SDS-PAGE and in various mammalian tissue sections. By directly staining SDS-PAGE gels with fluorescently labeled CMPs, both intact (type I, II, and IV) and MMP-1 cleaved collagen (type I) chains as well as complement factor C1q were detected. Collagen bands containing as little as 5 ng were optically visualized, while no staining was observed for fibronectin, laminin, and a collection of proteins from mammalian cell lysate. The CMP was unable to stain collagen-like bacterial protein, which contains numerous charged amino acids that are believed to stabilize triple helix in place of Hyp. We also show that fluorescently labeled CMPs can specifically visualize collagens in fixed tissue sections (e.g., skin, cornea, and bone) more effectively than anticollagen I antibody, and allow facile identification of pathologic conditions in fibrotic liver tissues.

116. Li, Y., Ho, D., Meng, H., Chan, T.R., An, B., Yu, H., Brodsky, B., Jun, A., and Yu, M. (2013) Direct detection of collagenous proteins by fluorescently labeled collagen mimetic peptides. Bioconjugate Chemistry, 16 January 2013; 24(1):9-16. DOI: 10.1039/C2IB20229H

Although fibrous collagens are major structural components of extracellular matrix in mammals, collagen overproduction is associated with many human diseases including cancers and fibrosis. Collagen is typically identified in biomedical research by western blot and immunohistochemistry; however anti-collagen antibodies employed in these analyses are difficult to prepare and their affinities to collagen can diminish if collagen becomes denatured during analyses. Previously, we discovered that single-stranded collagen mimetic peptides [CMPs, sequence: (GlyProHyp)9] can bind to denatured collagen chains by triple helix hybridization. Here we present collagen-specific staining methods using simple CMPs conjugated to common fluorophores (e.g. carboxyfluorescein), which allow direct detection of collagens and collagen-like proteins in SDS-PAGE and in various mammalian tissue sections. By directly staining SDS-PAGE gels with fluorescently labeled CMPs, both intact (type I, II, and IV) and MMP-1 cleaved collagen (type I) chains as well as complement factor C1q were detected. Collagen bands containing as little as 5 ng were optically visualized while no staining was observed for fibronectin, laminin, and a collection of proteins from mammalian cell lysate. The CMP was unable to stain collagen-like bacterial protein which contains numerous charged amino acids that are believed to stabilize triple helix in place of Hyp. We also show that fluorescently labeled CMPs can specifically visualize collagens in fixed tissue sections (e.g., skin, cornea, and bone) more effectively than anti-collagen I antibody, and allow facile identification of pathologic conditions in fibrotic liver tissues.

115. Wang, Y., Toh, Y.C., Li, Q., Nugraha, B., Zheng, B., Lu, T.B., Gao, Y., Ng, M.M.K., and Yu, H. (2013) Mechanical compaction directly modulates the dynamics of bile canaliculi formation. Integrative Biology, 5(2): 390 - 401. DOI: 10.1039/C2IB20229H

Homeostatic pressure-driven compaction is a ubiquitous mechanical force in multicellular organisms and is proposed to be important in the maintenance of multicellular tissue integrity and function. Previous cell-free biochemical models have demonstrated that there are cross-talks between compaction forces and tissue structural functions, such as cell–cell adhesion. However, its involvement in physiological tissue function has yet to be directly demonstrated. Here, we use the bile canaliculus (BC) as a physiological example of a multicellular functional structure in the liver, and employ a novel 3D microfluidic hepatocyte culture system to provide an unprecedented opportunity to experimentally modulate the compaction states of primary hepatocyte aggregates in a 3D physiological-mimicking environment. Mechanical compaction alters the physical attributes of the hepatocyte aggregates, including cell shape, cell packing density and cell–cell contact area, but does not impair the hepatocytes’ remodeling and functional capabilities. Characterization of structural and functional polarity shows that BC formation in compact hepatocyte aggregates is accelerated to as early as 12 hours post-seeding; whereas non-compact control requires 48 hours for functional BC formation. Further dynamic immunofluorescence imaging and gene expression profiling reveal that compaction accelerated BC formation is accompanied by changes in actin cytoskeleton remodeling dynamics and transcriptional levels of hepatic nuclear factor 4a and Annexin A2. Our report not only provides a novel strategy of modeling BC formation for in vitro hepatology research, but also shows a first instance that homeostatic pressure-driven compaction force is directly coupled to the higher-order multicellular functions.

114. Narmada, B.C., Chia, S.M., Tucker-Kellogg, L., Yu, H. (2013) HGF regulates the activation of TGF-β1 in rat hepatocytes and hepatic stellate cells. Journal of Cellular Physiology, February 2013; 228(2): 393-401. DOI: 10.1002/jcp.24143

Hepatocyte growth factor (HGF) ameliorates experimental liver fibrosis through many mechanisms, including degradation of accumulated collagen and decreased expression of fibrotic genes. Investigating an upstream mechanism in which HGF could decrease many fibrotic effectors, we asked whether HGF regulates activation of the fibrotic cytokine TGF-β1. Specifically, we tested whether HGF decreases the levels of active TGF-β1, and whether such decrease depends on the predominantly hepatocyte-secreted protease plasmin, and whether it depends on the TGF-β1 activator thrombospondin-1 (TSP-1). With hepatocyte monocultures, we found HGF-induced hepatocyte proliferation did increase total levels of plasmin, while decreasing gene expression of fibrotic markers (PAI-1, TGF-β1 & TIMP-2). With in vitro models of fibrotic liver (HSC-T6 hepatic stellate cells, or co-cultures of HSC-T6 and hepatocytes), we found high levels of fibrosis-associated proteins such as TSP-1, active TGF-β1, and Collagen I. HGF treatment on these fibrotic cultures stimulated plasmin levels; increased TSP-1 protein cleavage; and decreased the levels of active TGF-β1 and Collagen I. When plasmin was blocked by the inhibitor aprotinin, HGF could no longer decrease TGF-β1 activation and Collagen I. Meanwhile, the TSP-1-specific peptide inhibitor, LSKL, reduced TGF-β1 to the same level as in the HGF-treated cultures; combining LSKL and HGF treatments caused no further decrease, suggesting that HGF affects the TSP-1 dependent pathway of TGF-β1 activation. Therefore, HGF can decrease TGF-β1 activation and TGF-β1-dependent fibrotic markers, by stimulating hepatocytes to produce plasmin, and by antagonizing TSP-1-dependent activation of TGF-β1.

Earlier

113. Wang, X., Magalhães, R., Wu, Y., Wen, F., Gouk, S.S., Watson, P.F., Yu, H., and Kuleshova, L.L. (2012) Development of a modified vitrification strategy suitable for subsequent scale-up for hepatocyte preservation. Cryobiology, December 2012; 65(3): 289-300. DOI: 10.1016/j.cryobiol.2012.07.080

This work explores the design of a vitrification solution (VS) for scaled-up cryopreservation of hepatocytes, by adapting VSbasic (40% (v/v) ethylene glycol 0.6M sucrose, i.e. 7.17M ethylene glycol 0.6M sucrose), previously proven effective in vitrifying bioengineered constructs and stem cells. The initial section of the scale-up study involved the selection of non-penetrating additives to supplement VSbasic and increase the solution's total solute concentration. This involved a systematic approach with a step-by-step elimination of non-penetrating cryoprotectants, based on their effect on cells after long/short term exposures to high/low concentrations of the additives alone or in combinations, on the attachment ability of hepatocytes after exposure. At a second stage, hepatocyte suspension was vitrified and functions were assessed after continuous culture up to 5days. Results indicated Ficoll as the least toxic additive. Within 60min, the exposure of hepatocytes to a solution composed of 9% Ficoll+0.6M sucrose (10-3M Ficoll+0.6M sucrose) sustained attachment efficiency of 95%, similar to control. Furthermore, this additive did not cause any detriment to the attachment of these cells when supplementing the base vitrification solution VSbasic. The addition of 9% Ficoll, raised the total solute concentration to 74.06% (w/v) with a negligible 10-3M increase in molarity of the solution. This suggests main factor in inducing detriment to cells was the molar contribution of the additive. Vitrification protocol for scale-up condition sustained hepatocyte suspension attachment efficiency and albumin production. We conclude that although established approach will permit scaling-up of vitrification of hepatocyte suspension, vitrification of hepatocytes which are attached prior to vitrification is more effective by comparison.

112. Zheng, D., Limmon, G., Yin, L., Chow, V.T.K., Yu, H., and Chen, J. (2012) Regeneration of Alveolar Type I and II Cells from Scgb1a1-Expressing Cells Following Severe Pulmonary Damage Induced by Bleomycin and Influenza. PLoS One, 31 October 2012; 7(10): e48451. DOI: 10.1371/journal.pone.0048451

The lung comprises an extensive surface of epithelia constantly exposed to environmental insults. Maintaining the integrity of the alveolar epithelia is critical for lung function and gaseous exchange. However, following severe pulmonary damage, what progenitor cells give rise to alveolar type I and II cells during the regeneration of alveolar epithelia has not been fully determined. In this study, we have investigated this issue by using transgenic mice in which Scgb1a1-expressing cells and their progeny can be genetically labeled with EGFP. We show that following severe alveolar damage induced either by bleomycin or by infection with influenza virus, the majority of the newly generated alveolar type II cells in the damaged parenchyma were labeled with EGFP. A large proportion of EGFP-expressing type I cells were also observed among the type II cells. These findings strongly suggest that Scgb1a1-expressing cells, most likely Clara cells, are a major cell type that gives rise to alveolar type I and II cells during the regeneration of alveolar epithelia in response to severe pulmonary damage in mice.

111. Xia, L., Arooz, T., Zhang, S., Tuo, X., Xiao, G., Susanto, T.A.K., Sundararajan, J., Cheng, T., Kang, Y., Poh, H.J., Leo, H. L., and Yu, H. (2012) Hepatocyte function within a stacked double sandwich culture plate cylindrical bioreactor bioartificial liver system. Biomaterials, November 2012; 33(32): 7925-7932. DOI: 10.1016/j.biomaterials.2012.06.078

Bioartificialliver (BAL) system is promising as an alternative treatment for liver failure. We have developed abioreactor with stackedsandwichcultureplates for the application of BAL. This bioreactor design addresses some of the persistent problems in flat-bed bioreactors through increasing cell packing capacity, eliminating dead flow, regulating shear stress, and facilitating the scalability of the bioreactor unit. The bioreactor contained a stack of twelve double-sandwich-cultureplates, allowing 100 million hepatocytes to be housed in a single cylindrical bioreactor unit (7 cm of height and 5.5 cm of inner diameter). The serial flow perfusion through the bioreactor increased cell-fluid contact area for effective mass exchange. With the optimal perfusion flow rate, shear stress was minimized to achieve high and uniform cell viabilities across different plates in the bioreactor. Our results demonstrated that hepatocytes cultured in the bioreactor could re-establish cell polarity and maintain liver-specific functions (e.g. albumin and urea synthesis, phase I&II metabolism functions) for seven days. The single bioreactor unit can be readily scaled up to house adequate number of functional hepatocytes for BAL development.

110. Venkatraman, L., Chia, S.M., Narmada, B.C., White, J., Bhowmick, S.S., Dewey, C.F.Jr., So, P.T., Tucker-Kellogg, L., and Yu, H. (2012) Plasmin Triggers a Switch-like Decrease in Thrombospondin-Dependent Activation of TGF-β1. Biophysical Journal, 5 September 2012; 103(5): 1060-1068. DOI: 10.1016/j.bpj.2012.06.050

Transforming growth factor-β1 (TGF-β1) is a potent regulator of extracellular matrix production, wound healing, differentiation, and immune response, and is implicated in the progression of fibrotic diseases and cancer. Extracellular activation of TGF-β1 from its latent form provides spatiotemporal control over TGF-β1 signaling, but the current understanding of TGF-β1 activation does not emphasize cross talk between activators. Plasmin (PLS) and thrombospondin-1 (TSP1) have been studied individually as activators of TGF-β1, and in this work we used a systems-level approach with mathematical modeling and in vitro experiments to study the interplay between PLS and TSP1 in TGF-β1 activation. Simulations and steady-state analysis predicted a switch-like bistable transition between two levels of active TGF-β1, with an inverse correlation between PLS and TSP1. In particular, the model predicted that increasing PLS breaks a TSP1-TGF-β1 positive feedback loop and causes an unexpected net decrease in TGF-β1 activation. To test these predictions in vitro, we treated rat hepatocytes and hepatic stellate cells with PLS, which caused proteolytic cleavage of TSP1 and decreased activation of TGF-β1. The TGF-β1 activation levels showed a cooperative dose response, and a test of hysteresis in the cocultured cells validated that TGF-β1 activation is bistable. We conclude that switch-like behavior arises from natural competition between two distinct modes of TGF-β1 activation: a TSP1-mediated mode of high activation and a PLS-mediated mode of low activation. This switch suggests an explanation for the unexpected effects of the plasminogen activation system on TGF-β1 in fibrotic diseases in vivo, as well as novel prognostic and therapeutic approaches for diseases with TGF-β dysregulation.

109. Huang, T., Wang, J., Cai, Y.D., Yu, H., and Chou, K.C. (2012) Hepatitis C Virus Networks Based Classification of Hepatocellular Cirrhosis and Carcinoma. PLoS One, April 2012; 7(4): e34460. DOI: 10.1371/journal.pone.0034460

Hepatitis C virus (HCV) is a main risk factor for the liver cirrhosis and hepatocellular carcinoma, particularly to those patients with chronic liver disease or injury. The similar etiology leads to a high correlation of the patients suffering from the disease of liver cirrhosis with those suffering from the disease of hepatocellular carcinoma. However, the biological mechanism for the relationship between these two kinds of diseases is not clear. The present study was initiated in an attempt to investigate into the HCV infection protein network, in hopes to find good biomarkers for diagnosing the two diseases as well as gain insights into their progression mechanisms. To realize this, two potential biomarker pools were defined: (i) the target genes of HCV, and (ii) the between genes on the shortest paths among the target genes of HCV. Meanwhile, a predictor was developed for identifying the liver tissue samples among the following three categories: (i) normal, (ii) cirrhosis, and (iii) hepatocellular carcinoma. Interestingly, it was observed that the identification accuracy was higher with the tissue samples defined by extracting the features from the 2nd biomarker pool than that with the samples defined based on the 1st biomarker pool. The identification accuracy by the jackknife validation for the between-genes approach was 0.960, indicating that the novel approach holds a quite promising potential in helping find effective biomarkers for diagnosing the liver cirrhosis disease and the hepatocellular carcinoma disease. It may also provide useful insights for in-depth study of the biological mechanisms of HCV-induced cirrhosis and hepatocellular carcinoma.

108. Choudhury, D., van Noort, D., Iliescu, C., Zheng, B., Poon, K.L., Korzh, S., Korzh, V., and Yu, H. (2011) Fish and Chips: a microfluidic perfusion platform for monitoring zebrafish development. Lab on a Chip, 7 February 2012; 12(5): 892-900. DOI: 10.1039/C1LC20351G

We have developed a multi-channel microfluidic perfusion platform for culturing zebrafish embryos and capturing live images of various tissues and organs inside the embryo. The Fish and Chips was micro-fabricated in silicon and glass for reproducibility and accuracy of the microfluidic structure. The microfluidic platform consists of three parts: a microfluidic gradient generator, a row of eight fish tanks, in which the fish embryos are individually placed, and eight output channels. The fluidic gradient generator supports dose-dependent drug and chemical studies. A unique perfusion system ensures a uniform and constant flow of media to the fish tank while the wastes are efficiently removed. The fish tanks restrict the embryo movements, except rotationally, for live imaging of internal tissues and organs. The embryos showed developmental abnormalities under the influence of the drug valproic acid (VPA).

107. Xia, L., Sakban, R.B., Qu, Y., Hong, X., Zhang, W., Nugraha, B., Tong, W.H., Ananthanarayanan, A., Zheng, B., Chau, I.Y.Y., Jia, R., McMillian, M., Silva, J., Dallas, S., and Yu, H. (2012) Tethered spheroids as an in vitro hepatocyte model for drug safety screening. Biomaterials, March 2012; 33(7): 2165-2176. DOI: 10.1016/j.biomaterials.2011.12.006

Hepatocyte spheroids mimic many in vivo liver-tissue phenotypes but increase in size during extended culture which limits their application in drug testing applications. We have developed an improved hepatocyte 3D spheroid model, named "tethered spheroids", on RGD and galactoseconjugated membranes using an optimized hybrid ratio of the two bioactive ligands. Cells in the spheroid configuration maintained 3D morphology and uncompromised differentiated hepatocyte functions (urea and albumin production), while the spheroid bottom was firmly tethered to the substratum maintaining the spheroid size in multi-well plates. The oblate shape of the tethered spheroids, with an average height of 32 μm, ensured efficient nutrient, oxygen and drug access to all the cells within the spheroid structure. Cytochrome P450 induction by prototypical inducers was demonstrated in the tethered spheroids and was comparable or better than that observed with hepatocyte sandwich cultures. These data suggested that tethered 3D hepatocyte spheroids may be an ideal alternative to 2D hepatocyte culture models for drug safety applications.

106. Margadant, F., Chew, L.L., Hu, X., Yu, H., Bate, N., Zhang, X., and Sheetz, M. (2011) Mechanotransduction in Vivo by Repeated Talin Stretch-Relaxation Events Depends Upon Vinculin. PLoS Biology, 20 December 2011; 9(12): e1001223. DOI: 10.1371/journal.pbio.1001223

Mechanotransduction is a critical function for cells, in terms of cell viability, shaping of tissues, and cellular behavior. In vitro, cellular level forces can stretch adhesion proteins that link extracellular matrix to the actin cytoskeleton exposing hidden binding sites. However, there is no evidence that in vivo forces produce significant in vivo stretching to cause domain unfolding. We now report that the adhesion protein, talin, is repeatedly stretched by 100–350 nm in vivo by myosin contraction of actin filaments. Using a functional EGFP-N-Talin1-C-mCherry to measure the length of single talin molecules, we observed that the C-terminal mCherry was normally displaced in the direction of actin flow by 90 to >250 nm from N-EGFP but only by 50–60 nm (talin’s length in vitro) after myosin inhibition. Individual talin molecules transiently stretched and relaxed. Peripheral, multimolecular adhesions had green outside and red proximal edges. They also exhibited transient, myosin-dependent stretching of 50–350 nm for 6–16 s; however, expression of the talin-binding head of vinculin increased stretching to about 400 nm and suppressed dynamics. We suggest that rearward moving actin filaments bind, stretch, and release talin in multiple, stochastic stick-slip cycles and that multiple vinculin binding and release cycles integrate pulling on matrices into biochemical signals.

105. Magalhães, R., Nugraha, B., Pervaizb, S., Yu, H., Kuleshova, L.L. (2012) Influence of cell culture configuration on the post-cryopreservation viability of primary rat hepatocytes. Biomaterials, January 2012; 33(3): 829-836. DOI: 10.1016/j.biomaterials.2011.10.015

Cryopreservation has been identified as a necessary barrier to overcome in the production of tissue engineered products for clinical application. Liver engineering and bioartificial liver assisting devices are on the forefront of tissue engineering research due to its high demand and clinical potential. In this study we propose that the cryopreservation of primary mammalian hepatocytes yields better results when these cells are in a tissue-like culture configuration since cell attachment is essential for cell survival in this cell type. We used two different tissue-engineered culture configurations: monolayers and spheroid culture; and two different concepts of cryopreservation, namely vitrification and freezing. Cell suspensions were also cryopreserved using both approaches and results were compared to the engineered cultures. Both engineered configurations and suspension were cryopreserved using both conventional freezing (cooling at 1 C/minute using 10% DMSO in foetal calf serum) and vitrification (using 40% ethylene glycol 0.6 M sucrose supplemented with 9% Ficoll). These two approaches differ on the degree of mechanical stress they inflict on the material to be cryopreserved. The maintenance of cell-to-cell and the integrity of the actin cytoskeleton were assessed using scanning electron microscopy and immunohistochemistry respectively. Results showed that while there was no significant difference between the degree of integrity shown between vitrified and control engineered cultures, the same did not happen to the frozen engineered constructs. The disruption of the cytoskeletal structure correlated with increased levels of apoptotic markers. With cryopreserved suspensions there was evidence of disruption of the cytoskeletal structure. This study concluded that cell-to-cell contact is beneficial in the maintenance of viability post-cryopreservation and that the vitrification approach was far superior to those of conventional freezing when applied to 2D and 3D hepatocyte based engineered cultures.

104. Lin, J., Lu, F., Zheng, W., Xu, S., Tai, D., Yu, H., and Huang, Z. (2011) Assessment of liver steatosis and fibrosis in rats using integrated coherent anti-Stokes Raman scattering and multiphoton imaging technique. Journal of Biomedical Optics, 10 November 2011; 16:116024. DOI: 10.1117/1.3655353

We report the implementation of a unique integrated coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), and two-photon excitation fluorescence (TPEF) microscopy imaging technique developed for label-free monitoring of the progression of liver steatosis and fibrosis generated in a bile duct ligation (BDL) rat model. Among the 21 adult rats used in this study, 18 rats were performed with BDL surgery and sacrificed each week from weeks 1 to 6 (n = 3 per week), respectively; whereas 3 rats as control were sacrificed at week 0. Colocalized imaging of the aggregated hepatic fats, collagen fibrils, and hepatocyte morphologies in liver tissue is realized by using the integrated CARS, SHG, and TPEF technique. The results show that there are significant accumulations of hepatic lipid droplets and collagen fibrils associated with severe hepatocyte necrosis in BDL rat liver as compared to a normal liver tissue. The volume of normal hepatocytes keeps decreasing and the fiber collagen content in BDL rat liver follows a growing trend until week 6; whereas the hepatic fat content reaches a maximum in week 4 and then appears to stop growing in week 6, indicating that liver steatosis and fibrosis induced in a BDL rat liver model may develop at different rates. This work demonstrates that the integrated CARS and multiphoton microscopy imaging technique has the potential to provide an effective means for early diagnosis and detection of liver steatosis and fibrosis without labeling.

103. Toh, Y.C., Blagovic, K., Yu, H., and Voldman, J. (2011) Spatially organized in vitro models instruct asymmetric stem cell differentiation. Integrative Biology, 1 December 2011; 3(12): 1179-1187. DOI: 10.1039/c1ib00113b.

Understanding developmental biology requires knowledge of both the environmental factors regulating stem cell differentiation, which are increasingly being defined, and their spatial organization within a structurally heterogeneous niche, which is still largely unknown. Here we introduce spatially organized stem cell developmental models to interrogate the role of space in fate specification. Specifically, we developed Differential Environmental Spatial Patterning (*ESP) to organize different microenvironments around single embryonic stem cell (ESC) colonies via sequential micropatterning. We first used *ESP to decouple and understand the roles of cell organization and niche organization on ESCs deciding between self-renewal and differentiation fate choices. We then approximated in vitro an embryonic developmental step, specifically proximal-distal (PD) patterning of the mouse epiblast at pre-gastrulation, by spatially organizing two extraembryonic environments around ESCs, demonstrating that spatial organization of these three cell types is sufficient for PD patterns to form in vitro.

102. Zheng, B., Tan, L., Mo, X., Yu, W., Wang, Y., Tucker-Kellogg, L., Welsch, R.E., So, P.T.C., and Yu, H. (2011) Predicting in vivo anti-hepatofibrotic drug efficacy based on in vitro high-content analysis. PLoS ONE, 6(11):e26230. DOI: 10.1371/journal.pone.0026230

Background/Aims: Many anti-fibrotic drugs with high in vitro efficacies fail to produce significant effects in vivo. The aim of this work is to use a statistical approach to design a numerical predictor that correlates better with in vivo outcomes.
Methods: High content analysis (HCA) was performed with 49 drugs on hepatic stellate cells (HSCs) LX-2 stained with 10 fibrotic markers. ~ 0.3 billion feature values from all cells in >150,000 images were quantified to reflect the drug effects. A systematic literature search on the in vivo effects of all 49 drugs on hepatofibrotic rats yields 28 papers with histological scores. The in vivo and in vitro datasets were used to compute a single efficacy predictor (Epredict).
Results: We used in vivo data from one context (CCl4 rats with drug treatments) to optimize the computation of Epredict. This optimized relationship was independently validated using in vivo data from two different contexts (treatment of DMN rats and prevention of CCl4 induction). A linear in vitro-in vivo correlation was consistently observed in all the three contexts. We used Epredict values to cluster drugs according to efficacy; and found that high-efficacy drugs tended to target proliferation, apoptosis and contractility of HSCs.
Conclusions: The Epredict statistic, based on a prioritized combination of in vitro features, provides a better correlation between in vitro and in vivo drug response than any of the traditional in vitro markers considered.

101. Venkatraman, L., Li, H., Dewey, C.F., White, J.K., Bhowmick, S.S., Yu, H. and Tucker-Kellogg, L. (2011) Steady states and dynamics of urokinase-mediated plasmin activation in silio and in vitro. Biophysical Journal, 19 October 2011; 101(8): 1825-1834.. DOI: 10.1016/j.bpj.2011.08.054

Plasmin (PLS) and urokinase-type plasminogen activator (UPA) are ubiquitous proteases that regulate the extracellular environment. Although they are secreted in inactive forms, they can activate each other through proteolytic cleavage. This mutual interplay creates the potential for complex dynamics, which we investigated using mathematical modeling and in vitro experiments. We constructed ordinary differential equations to model the conversion of precursor plasminogen into active PLS, and precursor urokinase (scUPA) into active urokinase (tcUPA). Although neither PLS nor UPA exhibits allosteric cooperativity, modeling showed that cooperativity occurred at the system level because of substrate competition. Computational simulations and bifurcation analysis predicted that the system would be bistable over a range of parameters for cooperativity and positive feedback. Cell-free experiments with recombinant proteins tested key predictions of the model. PLS activation in response to scUPA stimulus was found to be cooperative in vitro. Finally, bistability was demonstrated in vitro by the presence of two significantly different steady-state levels of PLS activation for the same levels of stimulus. We conclude that ultrasensitive, bistable activation of UPA-PLS is possible in the presence of substrate competition. An ultrasensitive threshold for activation of PLS and UPA would have ramifications for normal and disease processes, including angiogenesis, metastasis, wound healing, and fibrosis.

100. Nugraha, B., Hong, X., Mo, X., Tan, L., Zhang, W., Chan, P.M., Kang, C.H., Wang, Y., Beng, L.T., Sun, W., Choudhury, D., Robens, J.M., McMillian, M., Silvia, J., Dallas, S., Tan, C.H., Yue, Z., and Yu, H. (2011) Galactosylated cellulosic sponge for multi-well drug safety testing. Biomaterials, October 2011; 32(29): 6982-6994. DOI: 10.1016/j.biomaterials.2011.05.087

Hepatocyte spheroids can maintain mature differentiated functions, but collide to form bulkier structures when in extended culture. When the spheroid diameter exceeds 200 μm, cells in the inner core experience hypoxia and limited access to nutrients and drugs. Here we report the development of a thin galactosylated cellulosic sponge to culture hepatocytes in multi-well plates as 3D spheroids, and constrain them within a macroporous scaffold network to maintain spheroid size and prevent detachment. The hydrogel-based soft sponge conjugated with galactose provided suitable mechanical and chemical cues to support rapid formation of hepatocyte spheroids with a mature hepatocyte phenotype. The spheroids tethered in the sponge showed excellent maintenance of 3D cell morphology, cell–cell interaction, polarity, metabolic and transporter function and/or expression. For example, cytochrome P450 (CYP1A2, CYP2B2 and CYP3A2) activities were significantly elevated in spheroids exposed to β-naphthoflavone, phenobarbital, or pregnenolone-16α-carbonitrile, respectively. The sponge also exhibits minimal drug absorption compared to other commercially available scaffolds. As the cell seeding and culture protocols are similar to various high-throughput 2D cell-based assays, this platform is readily scalable and provides an alternative to current hepatocyte platforms used in drug safety testing applications.

99. Anene-Nzelu, C., Wang, Y., Yu, H., and Leo, H.L. (2011) Liver tissue model for drug toxicity screening. Journal of Mechanics in Medicine and Biology, 11(2): 369-390. DOI: 10.1142/S0219519411004083

Understanding the mechanisms involved in the biotransformation of new drugs and their toxicological implications is important for drug development. In this regard, a lot of effort has been put into research to recreate the liver tissue in the laboratory for the purpose of drug screening. This has also helped to minimize the use of laboratory animal and reduce incidence of post-market withdrawal of drugs. Despite the progress made so far, cell source remains a major limitation since primary human hepatocytes are scarce and the various cell alternatives do not express all the genes found in the normal liver. In terms of tissue construct, there is a current shift to 3D models since the cell–cell interactions found in the 3D configuration enhance the morphology and function of hepatocytes. Furthermore, the engineered tissue's performance can be optimized by cocultures, perfusion-based systems, and the use of scaffolds. Nanotechnology seems promising in the field of tissue engineering, as it has been proven that cell–matrix interactions at the nano level can influence greatly on the outcome of the tissue. The review explores the various cell sources, the 3D model, flow-based systems, cocultures, and nanoscaffolds use in hepatocytes in vitro drug testing.

98. Choudhury, D., Mo, X., Iliescu, C., Tan, L.L., Tong, W.H., and Yu, H. (2011) Exploitation of physical and chemical constraints for three-dimensional microtissue construction in microfluidics. Biomicrofluidics, 29 June 2011; 5(2): 022203. DOI: 10.1063/1.3593407

There are a plethora of approaches to construct microtissues as building blocks for the repair and regeneration of larger and complex tissues. Here we focus on various physical and chemical trapping methods for engineering three-dimensional microtissue constructs in microfluidic systems that recapitulate the in vivo tissue microstructures and functions. Advances in these in vitro tissue models have enabled various applications, including drug screening, disease or injury models, and cell-based biosensors. The future would see strides toward the mesoscale control of even finer tissue microstructures and the scaling of various designs for high throughput applications. These tools and knowledge will establish the foundation for precision engineering of complex tissues of the internal organs for biomedical applications.

97. Zhou, J., Bi, C., Chng, W.J., Cheong, L.L., Liu, S.C., Mahara, S., Tay, K.G., Zeng, Q., Li, J., Guo, K., Tan, C.P.B., Yu, H., Albert, D.H., and Chen, C.S. (2011) PRL-3, a metastasis associated tyrosine phosphatase, is involved in FLT3-ITD signaling and implicated in anti-AML therapy. PLoS ONE, May 2011; 6(5): e19798. DOI: 10.1371/journal.pone.0019798

Combination with other small molecule drugs represents a promising strategy to improve therapeutic efficacy of FLT3 inhibitors in the clinic. We demonstrated that combining ABT-869, a FLT3 inhibitor, with SAHA, a HDAC inhibitor, led to synergistic killing of the AML cells with FLT3 mutations and suppression of colony formation. We identified a core gene signature that is uniquely induced by the combination treatment in 2 different leukemia cell lines. Among these, we showed that downregulation of PTP4A3 (PRL-3) played a role in this synergism. PRL-3 is downstream of FLT3 signaling and ectopic expression of PRL-3 conferred therapeutic resistance through upregulation of STAT (signal transducers and activators of transcription pathway activity and anti-apoptotic Mcl-1 protein. PRL-3 interacts with HDAC4 and SAHA downregulates PRL-3 via a proteasome dependent pathway. In addition, PRL-3 protein was identified in 47% of AML cases, but was absent in myeloid cells in normal bone marrows. Our results suggest such combination therapies may significantly improve the therapeutic efficacy of FLT3 inhibitors. PRL-3 plays a potential pathological role in AML and it might be a useful therapeutic target in AML, and warrant clinical investigation.

96. Yu, H.D., Zhang, Z.Y., Win, K.Y., Yu, H., Chang, J.K.Y., Teoh, S.H., and Han, M.Y. (2011) Fabrication and Osteoregenerative Application of Composition-Tunable CaCO3/HA Composites. Journal of Materials Chemistry, 21(12): 4588-4592. DOI: 10.1039/c0jm04161k

A series of porous CaCO3/HA composites have been produced with an increased molar percentage of HA from 0, 10, 25, 50, 75 to 100%. The compression strength of the CaCO3/HA composites increases proportionally before reaching 50% and gradually levels off with a further increase up to 100%. The 50% CaCO3/HA composite, with a similar compression strength to natural bone, has been evaluated in vitro and in vivo, showing great potential for osteoregenerative applications. A three-step chemical process for preparing the CaCO3/HA composites has been revealed by time-dependent XRD analysis:(i) CaCO3 /CaO + CO2[, (ii) 10CaO + 6(NH4)2HPO4 /Ca10(PO4)6(OH)2 + 12NH3[ + 8H2O[, and (iii) CaO + CO2 /CaCO3/Ca(OH)2 + CO2 /CaCO3 + H2O. The initial formation of porous CaO by a fast release of CO2 at high temperature leads to the production of the porous CaCO3/HA composites. Such understanding may allow us to extend this solid-phase fabrication approach to various other porous composites with controlled compositions for promising applications.

95. Ananthanarayanan, A., Narmada, B.C., Mo, X., McMillian, M., and Yu, H. (2010) Purpose-driven biomaterials research in liver tissue engineering. Trends in Biotechnology, March 2011, 29(3):110-118. DOI: 10.1016/j.tibtech.2010.10.006

Bottom-up engineering of microscale tissue (“microtissue”) constructs to recapitulate partially the complex structure–function relationships of liver parenchyma has been realized through the development of sophisticated biomaterial scaffolds, liver-cell sources, and in vitro culture techniques. With regard to in vivo applications, the long-lived stem/progenitor cell constructs can improve cell engraftment, whereas the short-lived, but highly functional hepatocyte constructs stimulate host liver regeneration. With regard to in vitro applications, microtissue constructs are being adapted or custom-engineered into cell-based assays for testing acute, chronic and idiosyncratic toxicities of drugs or pathogens. Systems-level methods and computational models that represent quantitative relationships between biomaterial scaffolds, cells and microtissue constructs will further enable their rational design for optimal integration into specific biomedical applications.

94. Zhang, S., Tong, W.H., Zheng, B., Susanto, T.A.K., Xia, L., Zhang, C., Ananthanarayanan, A., Tuo, X., Sakban, R.B., Jia, R.R., Iliescu, C., Chai, K.H., McMillian, M., Shen, S., Leo, H.L., and Yu, H. (2011) A Robust High-Throughput Sandwich Cell-based Drug Screening Platform. Biomaterials, February 2011; 32(4):1229-1241. DOI: 10.1016/j.biomaterials.2010.09.064

Hepatotoxicity evaluation of pharmaceutical lead compounds in early stages of drug development has drawn increasing attention. Sandwiched hepatocytes exhibiting stable functions in culture represent a standard model for hepatotoxicity testing of drugs. We have developed a robust and high-throughput hepatotoxicity testing platform based on the sandwiched hepatocytes for drug screening. The platform involves a galactosylated microfabricated membrane sandwich to support cellular function through uniform and efficient mass transfer while protecting cells from excessive shear. Perfusion bioreactor further enhances mass transfer and cellular functions over long period; and hepatoctyes are readily transferred to 96-well plate for high throughput robotic liquid handling. The bioreactor design and perfusion flow rate are optimized by computational fluid dynamics simulation and experimentally. The cultured hepatocytes preserved 3D cell morphology, urea production and cytochrome p450 activity of the mature hepatocytes for 14 days. When the perfusion-cultured sandwich is transferred to 96-well plate for drug testing, the hepatocytes exhibited improved drug sensitivity and low variability in hepatotoxicity responses amongst cells transferred from different dates of perfusion culture. The platform enables robust high-throughput screening of drug candidates.

93. Kuleshova, L.L., Gouk, S.S., Magalhaes, R., Wen, F., Wu, Y.N., Dawe, G.S., and Yu, H. (2010) Progress of vitrification: From single cells to multiple objects. Cryobiology, December 2010; 61(3): 370. DOI:10.1016/j.cryobiol.2010.10.031

Vitrification achieves cryopreservation without the formation of ice crystals, and therefore benefits cells and tissues known to be vulnerable to ice damage during conventional freezing. It is widely reported that oocytes, stem cells and other sensitive cells are adversely affected by freezing. Vitrification offers an improvement in both survival and non-lethal cell injury. Several areas of life sciences can significantly benefit from development of vitrification strategy. Vitrification of single object is now very well understood. My success on development of vitrification strategy leading to the world-first live birth from a vitrified oocyte is the last step in the line. This was accomplished 100 years after vitrification as a concept was proposed in physics, namely in 1898. Advancement in the area leads to development of vitrification strategies for multiple objects.

92. Zhang, X., Tee, Y.H., Heng, J.K, Zhu, Y., Hu, X., Margadant, F., Ballestrem, C., Bershadsky, A., Griffiths, G. and Yu, H. (2010) Kinectin-mediated endoplasmic reticulum dynamics supports focal adhesion growth in the cellular lamella. Journal of Cell Science, November 2010; 123(22):3901-3912. DOI: 10.1242/jcs.069153

Focal adhesions (FAs) are important in controlling cell shape and motility, important processes that underlie a wide range of physiological functions. FA dynamics is regulated by cytoskeleton, motor proteins and small GTPases. Kinectin is an integral endoplasmic reticulum (ER) membrane protein that extends the ER along microtubules. Here, we investigated ER influence on FA dynamics within the cellular lamella by disrupting the kinectin-kinesin interaction via over-expressing the minimal kinectin-kinesin interaction domain on kinectin in cells. This perturbation resulted in a morphological change to a rounded cell shape and reduced cell spreading and migration. Immuno-fluorescence and live cell imaging demonstrated a kinectin-dependent ER extension into the cellular lamella and ER co-localisation with FAs within the cellular lamella. FRAP experiment showed that ER contact with FAs was accompanied by an increase in FA protein recruitment to FAs. Disrupting kinectin-kinesin interaction caused a reduction in FA protein recruitment to FAs. These highlight that the ER supports FA growth within the cellular lamella. Microtubules targeting to FAs is known to promote adhesion disassembly but ER contact increased FA size even in the presence of microtubules. Our results suggest a scenario that kinectin-kinesin interaction facilitates ER transport along microtubules to support FA growth.

91. Yue, Z., Wen, F., Gao, S., Ang, M.Y., Pallathadka, P.K., Liu, L., and Yu, H. (2010) Preparation of three-dimensional interconnected macroporous cellulosic hydrogels for soft tissue engineering. Biomaterials, November 2010; 31(32):8141-8152. DOI: 10.1016/j.biomaterials.2010.07.059

This work exploits the thermal responsive phase behavior of hydroxypropylcellulose to produce 3D interconnected macroporous hydrogels in aqueous environment. Hydroxypropylcellulose was modified with allyl isocyanate, and their temperature mediated phase behavior was studied as a function of degree of modification (DS). A derivative with a DS of 1.5 was selected for scaffold preparation. Its aqueous solutions were warmed up to trigger the formation of biphasic systems. Such state was then immobilized efficiently by gamma-ray irradiated crosslinking. Lyophilization of the crosslinked hydrogels yielded 3D macroporous sponges. The re-hydrated gels demonstrate a combination of interconnected macroporosity, high water content and mechanical integrity to soft tissues. Cytocompatibility was demonstrated among various cell types, and in vivo biocompatibility test showed minimal inflammatory response within 12 weeks’ subcutaneous implantation in mice. The potential applications of these macroporous hydrogels in tissue engineering are discussed.

90. Raja, A.M., Xu, S., Zhou, J., Tai, D.C.S., Chen, C.S., and Yu, H. (2010) Pulse-modulated second harmonic imaging microscope imaging quantitatively demonstrates marked increase of collagen in tumor after chemotherapy. Journal of Biomedical Optics, September/October 2010; 15(5): 056016. DOI: 10.1117/1.3497565

Pulse-modulated second harmonic imaging microscopes (PM-SHIMs) exhibit improved signal-to-noise ratio (SNR) over conventional SHIMs on sensitive imaging and quantification of weak collagen signals inside tissues. We quantify the spatial distribution of sparse collagen inside a xenograft model of human acute myeloid leukemia (AML) tumor specimens treated with a new drug against receptor tyrosine kinase (ABT-869), and observe a significant increase in collagen area percentage, collagen fiber length, fiber width, and fiber number after chemotherapy. This finding reveals new insights into tumor responses to chemotherapy and suggests caution in developing new drugs and therapeutic regimens against cancers.

89. He, Y., Kang, C.H., Xu, S., Tuo, X., Trasti, S.L., Tai, D.C.S., Raja, A.M., Peng, Q., So, P.T.C., Rajapakse, J.C., Welsch, R., and Yu, H. (2010) Towards Surface Quantification of Liver Fibrosis Progression. Journal of Biomedical Optics, September/October 2010; 15(5):056007. DOI: 10.1117/1.3490414

Monitoring liver fibrosis progression by liver biopsy is important for certain treatment decisions, but repeated biopsy is invasive. We envision redefinition or elimination of liver biopsy with surface scanning of the liver with minimally invasive optical methods. This would be possible only if the information contained on or near liver surfaces accurately reflects the liver fibrosis progression in the liver interior. In our study, we acquired the second-harmonic generation and two-photon excitation fluorescence microscopy images of liver tissues from bile duct-ligated rat model of liver fibrosis. We extracted morphology-based features, such as total collagen, collagen in bile duct areas, bile duct proliferation, and areas occupied by remnant hepatocytes, and defined the capsule and subcapsular regions on the liver surface based on image analysis of features. We discovered a strong correlation between the liver fibrosis progression on the anterior surface and interior in both liver lobes, where biopsy is typically obtained. The posterior surface exhibits less correlation with the rest of the liver. Therefore, scanning the anterior liver surface would obtain similar information to that obtained from biopsy for monitoring liver fibrosis progression.

88. Mo, X., Li, Q., Lui, L.W.Y., Zheng, B., Kang, C.H., Nugraha, B., Yue, Z., Jia, R.R., Fu, H.X., Choudhury, D., Arooz, T., Yan, J., Lim, C.T., Shen, S., Tan, C.H., and Yu, H. (2010) Rapid Construction of Mechanically-confined Multi-cellular Structures using Dendrimeric Intercellular Linker. Biomaterials, October 2010; 31(29):7455-7467. DOI: 10.10j.biomaterials.2010.06.020

Tissue constructs that mimic the in vivo cell-cell and cell-matrix interactions are especially useful for applications involving the cell- dense and matrix- poor internal organs. Rapid and precise arrangement of cells into functional tissue constructs remains a challenge in tissue engineering.We demonstrate rapid assembly of C3A cells into multi- cell structures using a dendrimeric intercellular linker. The linker is composed of oleyl- polyethylene glycol (PEG) derivatives conjugated to a 16 arms- polypropylenimine hexadecaamine (DAB) dendrimer. The positively charged multivalent dendrimer concentrates the linker onto the negatively charged cell surface to facilitate ef!cient insertion of the hydrophobic oleyl groups into the cellular membrane. Bringing linker- treated cells into close proximity to each other via mechanical means such as centrifugation and micromanipulation enables their rapid assembly into multi- cellular structures within minutes. The cells exhibit high levels of viability, proliferation, three- dimensional (3D) cell morphology and other functions in the constructs. We constructed defined multi- cellular structures such as rings, sheets or branching rods that can serve as potential tissue building blocks to be further assembled into complex 3D tissue constructs for biomedical applications.

87. Zhang, W., Tucker-Kellogg, L., Narmada, B.C., Venkatraman, L., Chang, S., Yin, L., Tan, N., White, J.K., Jia, R.R., Bhowmick, S.S., Shen, S., Dewey, C.F. Jr., and Yu, H. (2010) Cell-Delivery Therapeutics for Liver Regeneration, Advanced Drug Delivery Reviews, June 2010; 62(7-8):814-826. DOI: 10.1016/j.addr.2010.02.005

For acute, chronic, or hereditary diseases of the liver, cell transplantation therapies can stimulate liver regeneration or serve as a bridge until liver transplantation can be performed. Recently, fetal hepatocytes, stem cells, liver progenitor cells, or other primitive and proliferative cell types have been employed for cell transplantation therapies, in an effort to improve the survival, proliferation, and engraftment of the transplanted cells. Reviewing earlier studies, which achieved success by transplanting mature hepatocytes, we propose that there is a switch-like regulation of liver regeneration that changes state according to a stimulus threshold of extracellular influences such as cytokines, matrices and neighboring cells. Important determinants of a successful clinical outcome include sufficient quantities and functional levels of the transplanted cells (even for short periods to alter the environment), rather than just engraftment levels or survival durations of the exogenously transplanted cells. The relative importance of these determining factors will impact future choices of cell sources, delivery vehicles, and sites of cell transplantation to stimulate liver regeneration for patients with severe liver diseases.

86. Ong, S.M., Zhao, Z., Arooz, T., Zhao, D., Zhang, S., Du, T., Wasser, M., van Noort, D., and Yu, H. (2010) Engineering a scaffold-free 3D tumor model for in vitro drug penetration studies, Biomaterials, February 2010; 31(6): 1180-1190. DOI: 10.1016/j.biomaterials.2009.10.049

Three-dimensional (3D) in vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in vivo conditions. In cancer research, the multi-cellular tumor spheroid (MCTS) model is an established 3D cancer model that exhibits microenvironmental heterogeneity close to that of tumors in vivo. However, the established process of MCTS formation is time-consuming and often uncontrolled. Here, we report a method for engineering MCTS using a transient inter-cellular linker which facilitates cell-cell interaction. Using C3A cells (a hepatocellular carcinoma cell line) as a model, we formed linker-engineered spheroids which grew to a diameter of 250 mm in 7 days, as compared to 16 days using conventional non-adherent culture. Seven-day old linker-engineered spheroids exhibited characteristics of mature MCTS, including spheroid morphology, gene expression profile, cell-cell interaction, extracellular matrix secretion, proliferation and oxygen concentration gradients, and cellular functions. Linker-engineered spheroids also displayed a resistance to drug penetration similar to mature MCTS, with dose-dependent extracellular accumulation of the drug. The linker-engineered spheroids thus provide a reliable accelerated 3D in vitro tumor model for drug penetration studies.

85. Ni, M., Tong, W.H., Choudhury, D., Abdul Rahim, N.A., Iliescu, C., and Yu, H. (2009) Cell Culture on MEMS Platforms: A Review, International Journal of Molecular Sciences, special issues on “Biocompatibility of Materials”, 10(12): 5411-5441. DOI: 10.3390/ijms10125411

Microfabricated systems provide an excellent platform for the culture of cells, and are an extremely useful tool for the investigation of cellular responses to various stimuli. Advantages offered over traditional methods include cost-effectiveness, controllability, low volume, high resolution, and sensitivity. Both biocompatible and bioincompatible materials have been developed for use in these applications. Biocompatible materials such as PMMA or PLGA can be used directly for cell culture. However, for bioincompatible materials such as silicon or PDMS, additional steps need to be taken to render these materials more suitable for cell adhesion and maintenance. This review describes multiple surface modification strategies to improve the biocompatibility of MEMS materials. Basic concepts of cell-biomaterial interactions, such as protein adsorption and cell adhesion are covered. Finally, the applications of these MEMS materials in Tissue Engineering are presented.

84. Liu, P., Zhang, Y.W., Yu, H., Zhang, X., Cheng, Q.H., Lu, C., and Bonfield, W. (2009) Spreading of an anchorage-dependent cell on a selectively ligand-coated substrate mediated by receptor-ligand binding. Journal of Biomedical Materials Research Part A, December 2009; 91(3): 806-813. DOI: 10.1002/jbm.a.32258

A continuum model was proposed to simulate a biological cell adhering to a substrate surface domain with selectively coated ligands. Cell adhesion was modeled by a traction-separation law depending on the receptor-ligand distance and their densities. It was found that the obtained spreading patterns are consistent with published experimental data and that adhesion requires a nonuniform distribution of receptors at the spreading front with convex fronts requiring a much higher receptor density than concave fronts. Adhesion strength was also found to be more influential than adhesion energy on the spreading kinetics.

83. Zhang, C., Zhao, Z., Abdul Rahim, N.A., van Noort, D., and Yu, H. (2009) Towards a Human-on-Chip: Culturing Multiple Cell Types on a Chip with Compartmentalized Microenvironments. Lab on a chip, 21 November 2009; 9(22): 3185-3192. DOI: 10.1039/b915147h

We have developed a multi-channel 3D microfluidic cell culture system (multi-channel 3D-μFCCS) with compartmentalized microenvironments for potential application in human drug screening. To this end, four cell types (C3A, A549, HK-2 and HPA) were chosen to represent the liver, lung, kidney and the adipose tissue, respectively. Cellular functions were optimized by supplementing the common medium with growth factors. However, TGF-β1 was found to enhance A549 functions but inhibit C3A functions. Therefore, TGF-β1 was specifically controlled-released inside the A549 compartment by means of gelatin microspheres mixed with cells, thus creating a cell-specific microenvironment. The function of A549 cells was enhanced while the functions of C3A, HK-2 and HPA cells were uncompromised, demonstrating the limited cross-talk between cell culture compartments similar to the in vivo situation. Such a multi-channel 3D-μFCCS could be potentially used to supplement or even replace animal models in drug screening.

82. Xia, L., Ng, S., Han, R., Tuo, X., Xiao, G., Leo, H.L., Cheng, T., and Yu, H. (2009) Laminar-flow immediate-overlay hepatocyte sandwich perfusion system for drug hepatotoxicity testing. Biomaterials, October 2009; 30(30): 5927-5936. DOI: 10.1016/j.biomaterials.2009.07.022

Drug hepatotoxicity testing requires in vitro hepatocyte culture to maintain the long-term and stable liver specific functions. We developed a drug testing platform based on laminar-flow immediate-overlay hepatocyte sandwich perfusion culture. The immediate-overlay sandwich (collagen-coated porous polymeric membrane as top overlay) protects the cells and integrity of the top collagen matrix from the impact of flow. A bioreactor was designed that allowed proper control of shear stress and mass transfer. The culture parameters such as the optimal perfusion initiation time and flow rate were systematically and mechanistically determined. The optimized system could re-establish hepatocyte polarity to support biliary excretion and to maintain other liver specific functions, such as the biotransformation enzyme activities, for two weeks that extended the usable in vitro hepatocyte-based drug testing window. When the perfusion cultured hepatocytes from days 7 or 14 were used for drug testing, the APAP-induced hepatotoxicity measurements were more sensitive and consistent over time than the static culture control, enabling further exploitations in large-scale drug testing applications.

81. Magalhães, R., Anil Kumar, Pr., Wen, F., Zhao, X., Yu, H., and Kuleshova, L.L. (2009) The use of vitrification to preserve primary rat hepatocyte monolayer on collagen-coated poly (ethylene-terephthalate) surfaces for a hybrid liver support device. Biomaterials, September 2009; 30(25):4136-4142. DOI: 10.1016/j.biomaterials.2009.04.037

We developed a scaled-up procedure for vitrifying hepatocytes for hybrid liver support system applications. Hepatocyte monolayer cultured on collagen-coated polyethylene terephthalate (PET) discs constituted the basic module for a hybrid liver support system. Freshly isolated rat hepatocytes were seeded on collagen-coated PET discs with a diameter of 33mm at a density of 5x10(6)cells per disc, and were cultured for 24h before cryopreservation. The total duration of procedure starting from exposure to low concentrations of cryoprotectants up to cryostorage is 10min. Vitrification of the modules was achieved by using two vitrification solutions sequentially with first vitrification solution containing two cryoprotectants, ethylene glycol (EG) and sucrose, while second vitrification solution contained additionally polymer, Ficoll. Direct exposure to liquid nitrogen vapours was followed by immersion into liquid nitrogen. Recovery procedure for vitrified modules included their warming in 1m sucrose at temperature of 38-39 degrees C followed by subsequently washing in sucrose-based solutions of decreased concentration within 15min at room temperature. Viability, structural characteristics, and functions of cells were preserved by vitrification. Hepatocytes in the post-vitrified and warmed monolayer maintained differentiated hepatocyte characteristics both structurally and functionally. In average, protein synthesis measured as albumin production was 181.00+/-33.46ng/million cells and 166.10+/-28.11ng/million cells, for control and vitrified modules respectively. Urea production was, in average, 1.52+/-0.40ng/million cells and 1.36+/-0.31ng/million cells for a 7 day culture respectively, with no significant statistical difference between the control and vitrified modules.

80. Zhang, C., Chia, S.M., Ong, S.M., Zhang, S., Toh, Y.C., van Noort, D., and Yu, H. (2009) The Controlled Presentation of TGF-β1 to Hepatocytes in a 3D-Microfluidic Cell Culture System. Biomaterials, August 2009; 30(23-24): 3847-3853. DOI: 10.1016/j.biomaterials.2009.03.052

3D-microfluidic cell culture systems (3D-mFCCSs) support hepatocyte functions in vitro which can be further enhanced by controlled presentation of 100–200 pg/ml TGF-β1, thus mimicking the roles of supporting cells in co-cultures. Controlled presentation of TGF-β1 is achieved by either direct perfusion or in situ controlled release from gelatin microspheres immobilized in the 3D-mFCCS. Primary hepatocytes cultured for 7 days with the in situ controlled-released TGF-β1 exhibited up to four-fold higher albumin secretion and two-fold higher phase I/II enzymatic activities, significantly improving the sensitivity of hepatocytes to acetaminophen-mediated hepatotoxicity, compared to hepatocytes cultured with directly perfused TGF-β1 or without TGF-β1. The controlled presentation of TGF-β1 enhanced hepatocyte functions in microfluidic systems without the complications of co-cultures, allowing for simplifications in drug testing and other hepatocyte-based applications.

79. Tai, D.C.S., Tan, N., Xu, S., Kang, C.H., Chia, S.M., Cheng, C.L., Wee, A., Wei, C.L., Raja, A.M., Xiao, G., Chang, S., Rajapakse, J.C., So, P.T.C., Tang, H.H., Chen, C.S., and Yu, H. (2009) Fibro-C-Index: comprehensive, morphology-based quantification of liver fibrosis using second harmonic generation and two-photon microscopy. Journal of Biomedical Optics, July-August 2009; 14(4): 044013. DOI: 10.1117/1.3183811

We have developed a standardized, fully automated, quantification system for liver fibrosis assessment using second harmonic generation microscopy and a morphology-based quantification algorithm. Liver fibrosis is associated with an abnormal increase in collagen as a result of chronic liver diseases. Histo-pathological scoring is the most commonly used method for liver fibrosis assessment, where liver biopsy is stained and scored by experienced pathologists. Due to the intrinsic limited sensitivity and operator-dependent variations, there exist high inter- and intra-observer discrepancies. We have validated our quantification system, Fibro-C-Index, with a comprehensive animal study and demonstrated its potential application in clinical diagnosis to reduce inter- and intra-observer discrepancies.

78. Toh, Y.C., Lim, T.C., Tai, D., Xiao, G., van Noort, D., and Yu, H. (2009) A microfluidic 3D hepatocyte chip for drug toxicity testing. Lab on a chip, July 2009; 9(14): 2062-2035. DOI: 10.1039/b900912d

We developed a microfluidic 3D hepatocyte chip (3D HepaTox Chip) for in vitro drug toxicity testing to predict in vivo drug hepatotoxicity. The 3D HepaTox Chip is based on multiplexed microfluidic channels where a 3D microenvironment is engineered in each channel to maintain hepatocytes' synthetic and metabolic functions. The multiplexed channels allows for simultaneous administration of multiple drug doses to functional primary hepatocytes while an incorporated concentration gradient generator enables the in vitro dose-dependent drug responses to predict in vivo hepatotoxicity. The IC50 values of 5 model drugs derived from the dose-dependent on-chip testing correlate well with the reported in vivo LD50 values. The 3D HepaTox Chip can be integrated with on-chip sensors and actuators as the next generation cell-based on-chip drug testing platform.

77. Zhou, J., Bi, C., Janakaumara, J., Liu, S.C., Chng, W.J., Tay, K.G., Poon, L.F., Xie, Z., Senthilnathan, P., Yu, H., Glaser, K.B., Albert, D., Davidsen, S., and Chen, C.S. (2009) Enhanced activation of STAT pathways and overexpression of survivin confer resistance to FLT3 inhibitors and could be therapeutic targets in AML. Blood, 23 April 2009; 113(17): 4052-4062. DOI: 10.1182/blood-2008-05-156422

To further investigate potential mechanisms of resistance to FLT3 inhibitors, we developed a resistant cell line by long-term culture of MV4-11 cells with ABT-869, designated as MV4-11-R. Gene profiling reveals up-regulation of FLT3LG (FLT3 ligand) and BIRC5 (survivin), but down-regulation of SOCS1, SOCS2, and SOCS3 in MV4-11-R cells. Hypermethylation of these SOCS genes leads to their transcriptional silencing. Survivin is directly regulated by STAT3. Stimulation of the parental MV4-11 cells with FLT3 ligand increases the expression of survivin and phosphorylated protein STAT1, STAT3, STAT5. Targeting survivin by shRNA in MV4-11-R cells induces apoptosis and augments ABT-869-mediated cytotoxicity. Overexpression of survivin protects MV4-11 from apoptosis. Sub-toxic dose of indirubin derivative (IDR) E804 resensitize MV4-11-R to ABT-869 treatment by inhibiting STAT signaling activity and abolishing survivin expression. Combining IDR E804 with ABT-869 shows potent in vivo efficacy in the MV4-11-R xenograft model. Taken together, these results demonstrate that enhanced activation of STAT pathways and overexpression of survivin are important mechanisms of resistance to ABT-869, suggesting that the STAT pathways and survivin could be potential targets for reducing resistance developed in patients receiving FLT3 inhibitors.

76. Pan, X., Shi, X., Korzh, V., Yu, H., and Wohland, T. (2009) Line scan fluorescence correlation spectroscopy for three-dimensional microfluidic flow velocity measurements. Journal of Biomedical Optics, March-April 20009; 14(2): 024049. DOI: 10.1117/1.3094947

The flow direction of microfluidics in biological applications is not limited to two dimensions, but often extends to three dimensions. Currently there are optical methods available for the measurement of 3D microfluidic flow vectors, but with low spatial resolution. Line scan fluorescence correlation spectroscopy (FCS) was proposed to determine flow directions in 2D within microchannels and small blood vessels in our previous work (Pan et al, J. Biomed. Opt. 2007, 12(1), 014034). Importantly, its spatial resolution was demonstrated to be as good as 0.5 μm. In this paper, we extend line scan FCS to the third dimension for the characterization of 3D flow velocity vectors. The spatial resolution is close to the diffraction limit using scan length of 0.5 μm in all three dimensions. The feasibility of line scan FCS for 3D microfluidic flow was verified by the measurements in microchannels and small blood vessels of zebrafish embryos.

75. van Noort, D., Ong, S.M., Zhang, C., Zhang, S., Arooz, T., and Yu, H. (2009) Stem cells in microfluidics. Biotechnology Progress, January-February 2009; 25(1):52-60. DOI: 10.1002/btpr.171

With the introduction of microtechnology and microfluidic platforms for cell culture, stem cell research can be put into a new context. Inside microfluidics, microenvironments can be more precisely controlled and their influence on cell fate studied. Microfluidic devices can be made transparent and the cells monitored real time by imaging, using fluorescence markers to probe cell functions and cell fate. This article gives a perspective on the yet untapped utility of microfluidic devices for stem cell research. It will guide the biologists through some basic microtechnology and the application of microfluidics to cell research, as well as highlight to the engineers the cell culture capabilities of microfluidics.

74. Sun, W., Chang, S., Tai, D.C.S., Tan, N., Xiao, G., Tang, H., and Yu, H. (2008) Non-linear optical microscopy: use of second harmonic generation and two-photon microscopy for automated quantitative liver fibrosis studies. Journal of Biomedical Optics, Nov-Dec 2008; 13(6):064010. DOI: 10.1117/1.3041159

Liver fibrosis is associated with abnormal increase in extracellular matrix (ECM) in chronic liver diseases. Quantitative characterization of fibrillar collagen in intact tissue is essential for both fibrosis studies and clinical applications. Commonly used methods, histological staining followed by either semi-quantitative or computerized image analysis, have limited sensitivity, accuracy and operator-dependent variations. The fibrillar collagen in sinusoids of normal livers could be observed through second harmonic generation (SHG) microscopy. The two-photon excited fluorescence (TPEF) images, recorded simultaneously with SHG, clearly revealed the hepatocyte morphology. We have systematically optimized the parameters for the quantitative SHG/TPEF imaging of liver tissue and developed fully-automated image analysis algorithms to extract the information of collagen changes and cell necrosis. Subtle changes in the distribution and amount of collagen and cell morphology are quantitatively characterized in SHG/TPEF images. By comparing with traditional staining such as Masson’s trichrome and Sirius red, SHG/TPEF is a sensitive quantitative tool for automated collagen characterization in liver tissue. Our system allows for enhanced detection and quantification of sinusoidal collagen fibers in fibrosis research and clinical diagnostics.

73. Chen, Z.C., Ekaputra, A.K., Gauthaman, K., Adaikan, P.G., Yu, H., and Hutmacher, D.W. (2008) In vitro and in vivo analysis of co-electrospun scaffolds made of medical grade poly(epsilon-caprolactone) and porcine collagen. Journal of Biomaterials Science. Polymer edition, 19(5):693-707. DOI: 10.1163/156856208784089580

In this study, a nanofiber mesh made by co-electrospinning medical grade poly(epsilon-caprolactone) and collagen (mPCL/Col) was fabricated and studied. Its mechanical properties and characteristics were analyzed and compared to mPCL meshes. mPCL/Col meshes showed a reduction in strength but an increase in ductility when compared to PCL meshes. In vitro assays revealed that mPCL/Col supported the attachment and proliferation of smooth muscle cells on both sides of the mesh. In vivo studies in the corpus cavernosa of rabbits revealed that the mPCL/Col scaffold used in conjunction with autologous smooth muscle cells resulted in better integration with host tissue when compared to cell free scaffolds. On a cellular level preseeded scaffolds showed a minimized foreign body reaction.

72. Zhang, S., Xia, L., Kang, C.H., Xiao, G., Ong, S.M., Toh, Y.C., Leo, H.L., van Noort, D., Kan, S.H., Tang, H.H., and Yu, H. (2008) Microfabricated silicon nitride membranes for hepatocyte sandwich culture. Biomaterials, October 2008; 29(29):3993-4002. DOI: 10.1016/j.biomaterials.2008.06.024

We have developed a hepatocyte sandwich culture with improved mass transport properties based on ultra-thin microfabricated porous silicon nitride (Si(3)N(4)) membranes. The dimensions and uniformity of the membrane pores can be configurable, which confers more control over the mass transport. Instead of collagen gels used in conventional sandwich culture, we utilized galactose ligands immobilized on the Si(3)N(4) membranes to support hepatocyte attachment and function in the sandwich culture. Diffusion studies using FITC-dextrans confirmed that mass transport of the microfabricated Si(3)N(4) membrane based sandwich was significantly better than conventional collagen gel sandwich and can be configured by varying the porosity of the Si(3)N(4) membrane. Hepatocytes cultured in the microfabricated Si(3)N(4) membrane based sandwich culture exhibited earlier apical repolarization and biliary excretion, improved differentiated functions and enhanced drug sensitivity compared to hepatocytes cultured in a collagen gel sandwich. The Si(3)N(4) membrane based sandwich culture allows for a systematic optimization of the mass transport properties of hepatocyte culture by changing the pore size and inter-pore distance. This will enable more effective drug testing applications where optimal mass transport is required for hepatocyte function maintenance and drug accessibility.

71. Wen, F., Chang, S., Toh, Y.C., Arooz, T., Zhuo, L., Teoh, S. H. and Yu, H. (2008) Development of dual-compartment perfusion bioreactor for serial co-culture of hepatocytes and stellate cells in poly (lactic-co glycolic acid)-collagen scaffolds. Journal of Biomedical Materials Research: Part B - Applied Biomaterials, October 2008; 87(1):154-162. DOI: 10.1002/jbm.b.31086

An important challenge in liver tissue engineering is to overcome the rapid loss of hepatocyte functions. In vivo, hepatocytes are compact polyhedral cells with round nuclei; however, they readily loss many of their differentiated functions in vitro. To overcome this challenge, we have established a new perfusion bioreactor that consists of two compartments which enabled the serial co-culture of hepatocytes and hepatic stellate cells-T6 without direct contact between each other. Three dimensional scaffolds were utilized in the bioreactor as physical anchors for cells. The scaffolds consist of collagen grafted poly (lactic-co glycolic acid) micro-fibers and cross-linked collagen sponges between micro-fibers for additional cellular support and adhesion. The advantages of this new bioreactor are enabling cell culture in three dimensional organization and controlling the culture parameters of the supporting cells independently from the hepatocytes. The results showed that the hepatocytes exhibited much higher levels of the differentiated functions such as albumin secretion, urea synthesis and cytochrome P450 enzymatic activity when compared to the mono-culture system where hepatocytes alone were cultured. This perfusion bioreactor system has potential applications in the development of bioartificial liver devices or cell-based tissue constructs transplantation therapies.

70. Magalhaes, R., Wang, X.W., Gouk, S.S., Lee, K.H., Ten, C.M., Yu, H. and Kuleshova, L.L. (2008) Vitrification successfully preserves hepatocyte spheroids. Cell Transplantation, 17(7):813-828. DOI: 10.3727/096368908786516765

This is the first report on low-temperature preservation of self-assembled cell aggregates by vitrification, which is both a time- and cost-effective technology. We developed an effective protocol for vitrification (ice-free cryopreservation) of hepatocyte spheroids that employs rapid stepwise exposure to cryoprotectants (10.5 min) at room temperature and direct immersion into liquid nitrogen (−196°C). For this, three vitrification solutions (VS) were formulated and their effects on vitrified-warmed spheroids were examined. Cryopreservation using ethylene glycol (EG)-sucrose VS showed excellent preservation capability whereby highly preserved cell viability and integrity of vitrified spheroids were observed, through confocal and scanning electron microscopy imaging, when compared to untreated control. The metabolic functions of EG-sucrose VS-cryopreserved spheroids, as assessed by urea production and albumin secretion, were not significantly different from those of control within the same day of observation. In both the vitrification and control groups, albumin secretion was consistently high, ranging from 47.57 ± 14.39 to 70.38 ± 11.29 μg/106 cells and from 56.84 ± 14.48 to 71.79 ± 16.65 μg/106 cells, respectively, and urea production gradually increased through the culture period. The efficacy of vitrification procedure in preserving the functional ability of hepatocyte spheroids was not improved by introduction of a second penetrating cryoprotectant, 1,2-propanediol (PD). Spheroids cryopreserved with EG-PD-sucrose VS showed maintained cell viability; however, in continuous culture, levels of both metabolic functions were lower than those cryopreserved with EG-sucrose VS. EG-PD VS, in which nonpenetrating cryoprotectant (sucrose) was excluded, provided poor protection to spheroids during cryopreservation. This study demonstrated that sucrose plays an important role in the effective vitrification of self-assembled cell aggregates. In a broad view, the excellent results obtained suggest that the developed vitrification strategy, which is an alternative to freezing, may be effectively used as a platform technology in the field of cell transplantation.

69. Zhao, D., Ong, S.M., Yue, Z., Jiang, Z., Toh, Y.C., Khan, M., Shi, J., Tan, C.H., Chen, J.P., and Yu, H. (2008) Dendrimer Hydrazides as Multivalent Transient Inter-Cellular Linkers. Biomaterials, September 2008; 29(27):3693-3702. DOI: 10.1016/j.biomaterials.2008.05.019

Three-dimensional (3D) multi-cellular aggregates (MCAs), as a model scaffold-free tissue construct, are useful for engineering cell-dense and matrix-poor tissues for repair and regeneration applications. To facilitate rapid MCA formation with high degrees of linker consistency and performance, we synthesized a class of dendrimer hydrazides with 8, 16 and 32 arms that can react with the aldehyde on the modified cell surfaces to form MCAs. DAB-AM-16 hydrazide with 32 arms demonstrated the best cell aggregation ability as compared to the dendrimer hydrazides with fewer arms, facilitating MCA formation at lower linker concentrations, minimizing cytotoxicity. Characterization of the MCAs formed with 2 microm of DAB-AM-16 hydrazide indicated that the cells proliferated well, maintained 3D cell-cell interaction and 3D cell morphology even as the inter-cellular linker gradually disappeared from the cell surfaces. Cells cultured as MCAs also demonstrated improved cell functions than the cells cultured in 2D monolayer. The dendrimer hydrazides would be a class of consistent, economical, and high performance multivalent transient inter-cellular linkers useful in forming scaffold-free 3D tissue constructs for soft-tissue engineering and regenerative medicine.

68. Ong, S.M., Zhang, C., Toh, Y.C., Kim, S.H., Foo, H.L., Tan, C.H., van Noort, D., Park, S., and Yu, H. (2008) A gel-free 3D microfluidic cell culture system. Biomaterials, August 2008; 29(22):3237-3244. DOI: 10.1016/j.biomaterials.2008.04.022

3D microfluidic cell culture systems offer a biologically relevant model to conduct micro-scale mammalian cell-based research and applications. Various natural and synthetic hydrogels have been successfully incorporated into microfluidic systems to support mammalian cells in 3D. However, embedment of cells in hydrogels introduces operational complexity, potentially hinders mass transfer, and is not suitable for establishing cell-dense, ECM-poor constructs. We present here a gel-free method for seeding and culturing mammalian cells three-dimensionally in a microfluidic channel. A combination of transient inter-cellular polymeric linker and micro-fabricated pillar arrays was used for the in situ formation and immobilization of 3D multi-cellular aggregates in a microfluidic channel. 3D cellular constructs formed this way are relieved of hydrogel embedment for cellular support. Two mammalian cell lines (A549 and C3A) and a primary mammalian cell (bone marrow mesenchymal stem cells) were cultured in the gel-free 3D microfluidic cell culture system. The cells displayed 3D cellular morphology, cellular functions and differentiation capability, affirming the versatility of the system as a 3D cell perfusion culture platform for anchorage-dependent mammalian cells.

67. Zhou, J., Khng, J., Jasinghe, V.J., Bi, C., Neo, C.H., Pan, M., Poon, L.F., Xie, Z., Yu, H., Yeoh, A.E., Lu, Y., Glaser, K.B., Albert, D.H., Davidsen, S.K., and Chen, C.S. (2008) In vivo activity of ABT-869, a multi-target kinase inhibitor, against acute myeloid leukemia with wild-type FLT3 receptor. Leukemia Research, July 2008; 32(7):1091-1100. DOI: 10.1016/j.leukres.2007.11.025

Neoangiogenesis plays an important role in leukemogenesis. We investigated the in vivo anti-leukemic effect of ABT-869 against AML with wild-type FLT3 using RFP transfected HL60 cells with in vivo imaging technology on both the subcutaneous and systemic leukemia xenograft models. ABT-869 showed a five-fold inhibition of tumor growth in comparison with vehicle control. IHC analysis revealed that ABT-869 decreased p-VEGFR1, Ki-67 labeling index, VEGF and remarkably increased apoptotic cells in the xenograft models. ABT-869 also reduced the leukemia burden and prolonged survival. Our study supports the rationale for clinically testing an anti-angiogenesis agent in AML with wild-type FLT3.

66. Toh, Y.C., Zhang, J.Z., Khong, Y.M., Du, Y., Sun, W. and Yu, H. (2008) Integrating sensitive quantification of hepatic metabolic functions by capillary electrophoresis with laser-induced fluorescence detection. The Analyst, March 2008; 133(3):326-330. DOI: 10.1039/b716420c

"We report the establishment of capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection as a common analytical platform for sensitive quantification of both phase I and II metabolism in various hepatic in vitro models."

65. Du, Y., Han, R., Wen, F., Ng S.S.S., Xia, L., Wohland, T., Leo, H.L., and Yu, H. (2008) Synthetic sandwich culture of 3D hepatocyte monolayer. Biomaterials, January 2008; 29(3):290-301. DOI: 10.1016/j.biomaterials.2007.09.016

The sandwich culture of hepatocytes, between double layers of extra-cellular matrix (ECM), is a well-established in vitro model for reestablishing hepatic polarity and maintaining differentiated functions. Applications of the ECM-based sandwich culture are limited by the mass transfer barriers induced by the top gelled ECM layer, complex molecular composition of ECM with batch-to-batch variation and uncontrollable coating of the ECM double layers. We have addressed these limitations of the ECM-based sandwich culture by developing an ‘ECM-free’ synthetic sandwich culture, which is constructed by sandwiching a 3D hepatocyte monolayer between a glycine-arginine-glycine-aspatic acid-serine (GRGDS)-modified polyethylene terephthalate (PET) track-etched membrane (top support) and a galactosylated PET film (bottom substratum). The bioactive top support and bottom substratum in the synthetic sandwich culture substituted for the functionalities of the ECM in the ECM-based sandwich culture with further improvement in mass transfer and optimal material properties. The 3D hepatocyte monolayer in the synthetic sandwich culture exhibited a similar process of hepatic polarity formation, better cell–cell interaction and improved differentiated functions over 14-day culture compared to the hepatocytes in collagen sandwich culture. The novel 3D hepatocyte monolayer sandwich culture using bioactive synthetic materials may readily replace the ECM-based sandwich culture for liver tissue engineering applications, such as drug metabolism/toxicity testing and hepatocyte-based bioreactors.

64. Khong, Y.M., Zhang, J., Zhou, S., Cheung, C., Doberstein, K., Samper, V., and Yu, H. (2007) Novel intra-tissue perfusion culture of thick liver slices. Tissue Engineering, September 2007; 13(9):2345-2356. DOI: 10.1089/ten.2007.0040

Innovative scaffold fabrication, angiogenesis promotion, and dynamic tissue culture techniques have been utilized to improve delivery of media into the core of large tissue constructs in tissue engineering. We have developed here an intra-tissue perfusion (ITP) system, which incorporates an array of 7 lm–sized needles as a delivery conduit, to improve mass transfer into the core of thick liver tissues slices (>>300 lmmass transport limit). The ITP system improves the uniformity and distribution of media throughout the tissue, resulting in improved cell viability over the static-cultured controls. The ITP-cultured thick liver slices also exhibit improved phase I and phase II metabolic functions and albumin and urea synthetic functions after 3-day culture, which is the minimal period required by the FDA for studying drug–drug interaction. This ITP system can also be used for culturing other thick tissue constructs of larger dimensions for various in vitro and in vivo applications, including bridging integration of the in vitro cultured constructs into living host tissues.

63. Ong, S.M., He, L., Linh, N.T.T., Tee, Y.H., Arooz, T., Tang, G., Tan, C.H., and Yu, H. (2007) Transient Inter-Cellular Polymeric Linker. Biomaterials, September 2007; 28 (25): 3656-3667. DOI: 10.1016/j.biomaterials.2007.04.034

Three-dimensional (3D) tissue-engineered constructs with bio-mimicry cell–cell and cell–matrix interactions are useful in regenerative medicine. In cell-dense and matrix-poor tissues of the internal organs, cells support one another via cell–cell interactions, supplemented by small amount of the extra-cellular matrices (ECM) secreted by the cells. Here we connect HepG2 cells directly but transiently with inter-cellular polymeric linker to facilitate cell–cell interaction and aggregation. The linker consists of a non-toxic low molecular-weight polyethyleneimine (PEI) backbone conjugated with multiple hydrazide groups that can aggregate cells within 30 min by reacting with the aldehyde handles on the chemically modified cell-surface glycoproteins. The cells in the cellular aggregates proliferated; and maintained the cortical actin distribution of the 3D cell morphology while non-aggregated cells died over 7 days of suspension culture. The aggregates lost distinguishable cell–cell boundaries within 3 days; and the ECM fibers became visible around cells from day 3 onwards while the inter-cellular polymeric linker disappeared from the cell surfaces over time. The transient inter-cellular polymeric linker can be useful for forming 3D cellular and tissue constructs without bulk biomaterials or extensive network of engineered ECM for various applications.

62. Du, Y., Han, R., Ng, S., Ni, J., Sun, W., Wohland, T., Ong, S.H., Kuleshova, L., and Yu, H. (2007) Identification and Characterization of a Novel Prespheroid 3-Dimensional Hepatocyte Monolayer on Galactosylated Substratum. Tissue Engineering, July 2007; 13(7):1455-1468. DOI: 10.1089/ten.2006.0381

Three-dimensional (3D) hepatocyte spheroids mimicking the structural and functional characteristics of hepatocytes in vivo were self-assembled onto a galactosylated polyethylene terephthalate (PET) substratum, and the dynamic process of spheroid formation was investigated using time-lapse confocal microscopy. Hepatocytes cultured on this galactosylated substratum formed small cell-aggregates within 12 h, which gradually merged into ‘‘island-like’’ clusters at approximately 1 day and spread to form prespheroid monolayer within 2 days; the prespheroid monolayer was stretched to fold into compact and larger 3D spheroids after 3 days. We compared the expressions of F-actin (cytoskeleton), phosphorylated focal adhesion kinase (p-FAK, cell–substratum interactions) and E-cadherin (cell–cell interactions) during the dynamic process of 3D hepatocyte spheroid formation with the dynamic process of 2D hepatocyte monolayer formation on collagen substratum. Hepatocytes in the prespheroid monolayer stage exhibited the strongest cell–substratum interactions of all 4 stages during spheroid formation with cell–cell interactions and F-actin distribution comparable with those of the 3D hepatocyte spheroids. The prespheroid monolayer also exhibited better hepatocyte polarity (multidrug resistance protein 2) and tight junction (zonula occludens-1) formation, more-differentiated hepatocyte functions (albumin production and cytochrome P450 1 A activity), and higher sensitivity to hepatotoxicity than the conventional 2D hepatocyte monolayer. The transient prespheroid 3D monolayer could be stabilized on a hybrid glycine–arginine– glycine–aspartic acid–serine (GRGDS)/galactose-PET substratum for up to 1 week and destabilized to form 3D spheroids in excess soluble GRGDS peptide.

61. Kim, L., Toh, Y.C., Voldman, J., and Yu, H. (2007) A practical guide to microfluidic perfusion culture of adherent mammalian cells. Lab on a Chip, June 2007; 7:681-694. DOI: 10.1039/b704602b

Culturing cells at microscales allows control over microenvironmental cues, such as cell–cell and cell–matrix interactions; the potential to scale experiments; the use of small culture volumes; and the ability to integrate with microsystem technologies for on-chip experimentation. Microfluidic perfusion culture in particular allows controlled delivery and removal of soluble biochemical molecules in the extracellular microenvironment, and controlled application of mechanical forces exerted via fluid flow. There are many challenges to designing and operating a robust microfluidic perfusion culture system for routine culture of adherent mammalian cells. The current literature on microfluidic perfusion culture treats microfluidic design, device fabrication, cell culture, and micro-assays independently. Here we systematically present and discuss important design considerations in the context of the entire microfluidic perfusion culture system. These design considerations include the choice of materials, culture configurations, microfluidic network fabrication and micro-assays. We also present technical issues such as sterilization; seeding cells in both 2D and 3D configurations; and operating the system under optimized mass transport and shear stress conditions, free of air-bubbles. The integrative and systematic treatment of the microfluidic system design and fabrication, cell culture, and micro-assays provides novices with an effective starting point to build and operate a robust microfludic perfusion culture system for various applications.

60. Pan, X., Foo, W., Lim, W., Fok, M.H., Liu P., Yu, H., Maruyama, I., and Wohland, T. (2007) Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements. Review of Scientific Instruments, May 2007; 78(5):053711. DOI: 10.1063/1.2740053

A modified fluorescence correlation microscope (FCM) was built on a commercial confocal laser scanning microscope (CLSM) by adding two sensitive detectors to perform fluorescence correlation spectroscopy (FCS). A single pinhole for both imaging and spectroscopy and a simple slider switch between the two modes thus facilitate the accurate positioning of the FCS observation volume after the confocal image acquisition. Due to the use of a single pinhole for CLSM and FCS the identity of imaged and spectroscopically observed positions is guaranteed. The presented FCM system has the capability to position the FCS observation volume at any point within the inner 30% of the field of view without loss in performance and in the inner 60% of the field of view with changes of FCS parameters of less than 10%. A single pinhole scheme for spatial fluorescence cross correlation spectroscopy performed on the FCM system is proposed to determine microfluidic flow angles. To show the applicability and versatility of the system, we measured the translational diffusion coefficients on the upper and lower membranes of Chinese hamster ovary cells. Two-photon excitation FCS was also realized by coupling a pulsed Ti: sapphire laser into the microscope and used for flow direction characterization in microchannels.

59. Wu, Y., Yu, H., Chang, S., Magalhães, R., and Kuleshova, L.L. (2007) Vitreous cryopreservation of cell-biomaterial constructs involving encapsulated hepatocytes. Tissue Engineering, March 2007; 13(3):649-658. DOI: 10.1089/ten.2006.0075

We put forward a new strategy for cryopreservation, namely vitrification or ice-free preservation, of cell–biomaterial constructs for tissue-engineering applications. In this study, for a period of 6 days, we tested vitrified and control hepatocytes entrapped at 2 different cell densities (1.5 × 106 and 5 × 106 cells/mL) in 2 types of engineered collagen matrices (M- and G-collagen) as models to evaluate efficacy and universality of the developed vitrification method. The nature of collagens caused differences in capsule sizes (100-200 μm versus 350-450 μm). The developed method included rapid step-wise introduction of microencapsulated hepatocytes to vitrification solution (40v/v% ethylene glycol 0.6 M sucrose in medium) and their direct immersion in liquid nitrogen. Vitrification did not affect viability and functions of the microencapsulated hepatocytes, which exhibited trends similar to those of untreated controls in the decline of their functions and the rate of cell death during continuous culture, irrespective of physical and chemical properties of the biomaterial and cell density. For control and vitrification, the percentage of live cells varied from 80.3% ± 0.9% to 82.3% ± 1.4% in capsules formed by M-collagen, from 82.8% ± 1.1% to 85.0% ± 3.3% in capsules formed by G-collagen with cells entrapped at low density, and from 84.4% ± 1.3% to 86.8% ± 0.6% in capsules formed by G-collagen with cells entrapped at high density (p > 0.05). Within the same day, the maximum relative change in cell viability and functions between control and vitrification was 4% and 16%, respectively. The developed vitrification approach, which is an alternative to freezing, can be applied to other tissue-engineered constructs with comparable sizes, various cell numbers, and various properties of the biomaterials involved.

58. Toh, Y.C., Zhang, C., Zhang, J., Khong, Y.M., Chang, S., Samper, V.D., van Noort, D., Hutmacher, D.W., and Yu, H. (cover March issue 2007) A novel 3D mammalian-cell perfusion-culture system in micro-fluidic channels. Lab on a Chip, 7(3):302-309. DOI: 10.1039/b614872g

Mammalian cells cultured on 2D surfaces in microfluidic channels are increasingly used in drug development and biological research applications. These systems would have more biological or clinical relevance if the cells exhibit 3D phenotypes similar to the cells in vivo. We have developed a microfluidic channel based system that allows cells to be perfusion-cultured in 3D by supporting them with adequate 3D cell–cell and cell–matrix interactions. The maximal cell–cell interaction was achieved by perfusion-seeding cells through an array of micropillars; and 3D cell–matrix interactions were achieved by a polyelectrolyte complex coacervation process to form a thin layer of matrix conforming to the 3D cell shapes. Carcinoma cell lines (HepG2, MCF7), primary differentiated (hepatocytes) and primary progenitor cells (bone marrow mesenchymal stem cells) were perfusion-cultured for 72 hours to 1 week in the microfluidic channel, which preserved their 3D cyto-architecture and cell-specific functions or differentiation competence. This transparent 3D microfluidic channel-based cell culture system also allows direct optical monitoring of cellular events for a wide range of applications.

57. Foo, H.L., Taniguchi, A., Yu, H., Okano, T., and Teoh, S.H. (2007) Catalytic surface modification of roll-milled poly(ε-caprolactone) biaxially stretched to ultra-thin dimension. Materials Science and Engineering: C, March 2007; 27(2):299-303. DOI: 10.1016/j.msec.2006.03.011

A novel roll-milling polymer processing technique along with biaxial stretching was used to fabricate 10 μm thick poly(ε-caprolactone) films. A less invasive collagen surface modification was used, involving a reaction between corona-preactivated membranes and ferrous-containing acrylic acid solution at the low temperature of 42 °C. Successful modified films were characterized by Toluidine Blue O assay and X-ray photoelectron spectroscopy. Human umbilical vein endothelial cells also showed both higher proliferation rate and differentiated cobblestone morphology on these collagen-immobilized substrates.

56. Wen, F., Chang, S., Toh, Y.C., Teoh, S.H., and Yu, H. (2007) Development of poly (lactic-co-glycolic acid)-collagen scaffolds for tissue engineering. Materials Science and Engineering: C, March 2007; 27(2):285-292. DOI: 10.1016/j.msec.2006.05.007

Collagen as an important extra-cellular matrix (ECM) in many tissues is weakly antigenic and the structure of collagen sponges is highly porous with interconnected pores effective for cell infiltration and mass transfer of oxygen and nutrients. Its application as a scaffold is limited by poor mechanical strength and rapid biodegradation. In this paper, we attempt to graft hydrolyzed PLGA fiber surfaces with collagen by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS), and then embed these collagen-grafted PLGA fibers in collagen sponge to form a hybrid PLGA-collagen scaffold. For further stability, we cross-linked the collagen in the scaffold and used it in rat liver cell cultivation. The scaffold was characterized by mechanical micro-tester, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Results showed that (1) the scaffolds exhibited isotropic and interconnected porous structure; (2) the compression modulus of this scaffold was enhanced 50 fold compared to the collagen scaffolds. The cell attachment and cytotoxicity of this scaffold were studied. Cell attachment was improved remarkably and the cytotoxicity of the hybrid PLGA-collagen scaffold was lower than that of the un-grafted PLGA-collagen scaffolds using alamarBlue™ assay normalized to the DNA content in each scaffold. This new hybrid scaffold has potential applications for tissue engineering.

55. Pan, X., Yu, H., Shi, X., Korzh, V., and Wohland, T. (2007) Characterization of flow direction in microchannels and zebrafish blood vessels by scanning fluorescence correlation spectroscopy. Journal of Biomedical Optics, January-February 2007; 12 (1):014034. DOI: 10.1117/1.2435173

The investigation of flow profiles in microstructures and tissues by fluorescence correlation spectroscopy (FCS) has been a challenging topic in the past decade. Due to its inherent optical configuration, a circular focused laser beam, FCS is unable to resolve microfluidic flow directions. Earlier schemes reported the use of two laser beams or the use of nonsymmetrical laser foci to break the symmetry of the measurement system. This, however, is difficult to combine with confocal systems since it would require modifications that interfere with the imaging capabilities. We propose a method called linescan FCS to measure different flow angles in microchannels and tissues. This method is implemented on a combined laser scanning confocal microscopy (LSCM) and FCS system that enables uncompromised imaging and spectroscopy measurements. We demonstrate that by scanning the laser beam with a defined speed and direction we can measure flow direction with the current system at an optimal resolution of at least 3 microm. The combination system is assessed by measuring flow profiles in a microchannel with and without obstruction. To extend the technique to live tissue measurements we demonstrate that line-scan FCS can determine the flow direction in zebrafish small blood vessels in a label-free approach.

54. Schumacher, K., Khong, Y.M., Chang, S., Ni, J., Sun, W., and Yu,H. (2007) Perfusion culture improves the maintenance of cultured liver tissue slices. Tissue Engineering, January 2007; 13(1):197-205. DOI: 10.1089/ten.2006.0046

Cultured precision-cut liver tissue slices are useful for studying the metabolism and toxicity of xenobiotics in liver. They may also be used to investigate the behavior of and interaction between different cell types in an intact histo-architecture. Because cultured liver tissues undergo a loss of function and morphology because of their separation from the blood supply, we investigated changes in key protein marker expressions in parenchymal and non-parenchymal cells, as well as in the extracellular matrix (ECM) at different time points. We also compared conventional culture methods such as static and dynamic cultures with perfusion culture, which allows a continuous exchange of the culture medium. In conventional culture methods, the expression of vimentin and collagen type IV decreased after 5 h in the non-parenchymal cells and the ECM, respectively, whereas the hepatocyte nuclear factor 4 alpha (HNF4a) staining in the hepatocytes remained constant. In perfusion culture, on the other hand, vimentin, collagen type IV, and HNF4 a staining were clearly detectable after 5 h. The histo-architecture obtained from perfusion culture was also more compact than those obtained from conventional culture methods. After 24 h, only the perfusion cultured sample retained protein marker expression in all components of the liver tissue. Our results suggest that, to develop improved culture techniques for liver slices, changes at the early time-points should be taken into consideration. Our results also show that culture techniques that enable a continuous exchange of the culture medium seem to be superior to static or dynamic cultures in terms of maintaining the protein expression and the histo-architecture.

53. Ong, L.L, Lin, P.C. Zhang, X., Chia, S.M., and Yu, H. (2006) Kinectin-dependent assembly of translation elongation factor-1 complex on endoplasmic reticultum regulates protein synthesis. Journal of Biological Chemistry, 3 November 2006; 281(44):33621-33634. DOI: 10.1074/jbc.M607555200

Kinectin is an integral membrane protein with many isoforms primarily found on the endoplasmic reticulum. It has been found to bind kinesin, Rho GTPase and translation elongation factor-1 delta. None of the existing models for the quaternary organization of the elongation factor-1 complex in higher eukaryotes involves kinectin. We have investigated here the assembly of the elongation factor-1 complex onto endoplasmic reticulum via kinectin using in vitro and in vivo assays. We established that the entire elongation factor-1 complex can be anchored to endoplasmic reticulum via kinectin and the interacting partners are: kinectin binds EF-1δ which in turn binds EF-1γ but not EF-1β; EF-1γ binds EF-1δ and EF-1β but not kinectin. In vivo splice-blocking of the kinectin exons 36 and 37 produced kinectin lacking the EF-1δ binding domain, which disrupted the membrane localization of EF-1δ, EF-1γ and EF-1β on endoplasmic reticulum, similar to the disruptions seen with the over-expression of kinectin fragments containing the EF-1δ binding domain. The disruptions of the EF-1δ/kinectin interaction inhibited expression of membrane proteins but enhances synthesis of cytosolic proteins in vivo. These findings suggest that anchoring the elongation factor-1 complex onto endoplasmic reticulum via EF-1δ/kinectin interaction is important for regulating protein synthesis in eukaryotic cells.

52. Du, Y., Chia, S.M., Han, R., Chang, S., Tang, H., and Yu, H. (2006) 3D Hepatocyte monolayer on hybrid RGD/Galactose substratum. Biomaterials, November 2006; 27(33):5669-5680. DOI: 10.1016/j.biomaterials.2006.07.018

Hepatocyte-based applications such as xenobiotics metabolism and toxicity studies usually require hepatocytes anchoring onto flat substrata that support their functional maintenance. Conventional cell culture plates coated with natural matrices or synthetic ligands allow hepatocytes to adhere tightly as two-dimensional (2D) monolayer but these tightly anchored hepatocytes rapidly lose their differentiated functions. On galactosylated substrata, hepatocytes adhere loosely; and readily form three-dimensional (3D) spheroids that can maintain high levels of cellular functions. These spheroids detach easily from the substrata and exhibit poor mass transport properties unsuitable for many applications. Here, we have developed a hybrid RGD/galactose substratum based on polyethylene terephthalate film conjugated with both RGD peptide and galactose ligand to enhance cell adhesion and functions synergistically. Primary hepatocytes adhere effectively onto the transparent hybrid substratum in 96-well plates as monolayer while exhibiting high levels of liver-specific functions, morphology and cell–cell interactions typically seen in the 3D hepatocyte spheroids. The hepatocytes cultured onto the hybrid substratum also exhibit high levels of sensitivity to a model drug acetaminophen similar to the 3D hepatocyte spheroids. The monolayer of hepatocytes exhibiting the 3D cell behaviors on this flat hybrid substratum can be useful for various applications requiring both effective mass transfer and cellular support.

51. Pan, X., Aw, C., Du, Y., Yu, H., and Wohland, T. (2006) Characterization of poly(acrylic acid) diffusion dynamics on the grafted surface of poly(ethylene terephthalate) films by fluorescence correlation spectroscopy. Biophysical Review and Letters, October 2006; 1(4): 433-441. DOI: 10.1142/S1793048006000264

Poly(acrylic acid) (PAAc) is a commonly used polymer grafted on poly(ethylene terephthalate) films for the immobilization of bioactive molecules that directly interact with 19 living cells or tissues for the maintenance of their viability and functionality. The diffusion property of the grafted PAAc on the surface is a critical parameter related to 21 the density, length of polymer chains, and ionic strength of the solution. Fluorescence correlation spectroscopy (FCS) is able to measure the diffusion coefficient of fluorescent 23 particles in solution with single molecule sensitivity and specificity. It was used as an effective tool to detect diffusion dynamics of Atto 565 molecules, a good indicator for 25 viscosity of PAAc, in both aqueous polymer solutions and polymer grafted film surfaces immersed in solution. In this work we determine the polymer chain length under dif- 27 ferent polymerization conditions in solution and deduce the solution viscosity by FCS measurements of Atto 565 as tracer molecule. By using the same tracer on the grafted 29 polymer films we can infer the viscosity of these grafted layers under a variety of conditions, including the PAAc chain length, the UV exposure time during polymerization, 31 the ionic strength, and the pH value of the immersed solution.

C2. Zhu, J., Zhang, B., Yan, X., Lao, X., and Yu, H. (2006) Encapsulating hepatocytes with chitosan in physiological conditions. [In chinese]. Shengwu yixue gongchengxue zazhi/ Journal of biomedical engineering, October 2006; 23(5): 1070-1074. PMID:17121357

Prepared from 15.3% N-acetylated chitosan (FNC), half N-acetylated chitosan (HNC) possesses a good solubility in a weak basic solution, guaranteeing the formation of microcapsules by the coacervating reaction between HNC and methacrylic acid (MAA)-hydroxyethyl methacrylate (HEMA)-methyl methacrylate (MMA) (MAA-HEMA-MMA) terpolymer under physiological conditions. When hepatocytes were encapsulated in such 3-dimensional microenvironment, as compared to monolayer culture, cell functions, including P450 activity, urea production and albumin release, were well supported. The prepared microcapsules have good mechanical stability and permeability.

50. Toh, Y.C., Ng, S., Khong, Y.M., Zhang, X., Zhu, Y., Lin, P.C., Ten, C.M., Sun, W.X., Yu, H. (2006) Cellular responses to a nanofibrous environment. Nano Today, August 2006; 1(3):34-43. DOI: 10.1016/S1748-0132(06)70078-0

Cells respond profoundly to the mechanical rigidity and three-dimensional nanotopology of substrates, as well as the spatial and temporal arrangements of extracellular cues. We summarize the latest developments in probing and engineering biocompatible nanofibrous extracellular environments at the cell and molecular level for applications in tissue engineering and biological research. This will, in turn, guide further development of three-dimensional nanofibrous scaffolds in order to elicit specific cellular responses for relevant applications.

49. Ng, S., Han, R., Chang, S., Ni, J., Hunziker, W., Goryachev, A.B., Ong, S.H., and Yu, H. (2006) Improved hepatocyte excretory function by immediate presentation of polarity cues. Tissue Engineering, August 2006; 12(8):2181-2191. DOI: 10.1089/ten.2006.12.2181

Liver tissue constructs with excretory function are crucial to developing realistic hepatocyte models for engineering effective bioartificial liver-assisted devices and for modeling the in vivo tissue. Current hepatocyte in vitro models suffer from limited or inefficient hepatocyte repolarization, which results in poor removal of xenobiotics and other waste products from the cells. We hypothesized that the temporal and spatial presentation of the cell matrix and cell-cell contacts as polarity cues would be important to define the axis of polarization to improve the excretory function of hepatocytes. The spatial presentation of polarity cues can be best achieved with sandwich configuration. We improve the temporal presentation of polarity cues by introducing the collagen overlay immediately in synchrony with cell-cell contacts instead of after 24 h in conventional sandwich culture. We demonstrate that the immediate presentation of the collagen matrix overlay enhances the formation of apicobasolateral domains, tight junctions, and the recovery of the functional activity of 2 canalicular transporters, the multidrug resistance–associated protein (Mrp2) and P-glycoprotein (P-gp) at 48 h of culture, and enhances the albumin secretion, urea production, and 7-ethoxyresorufin-O-deethylation cytochrome P450 activities of hepatocytes over 14 days of culture as compared to the 24-h overlay controls. The improvement in the excretory function of hepatocytes for the removal of waste products deleterious to cells may improve the functional maintenance and the in vivo fidelity of tissue-engineered liver constructs.

48. Wong, S.C., Yu, H., and So, J.B. (2006) Detection of telomerase activity in gastric lavage fluid: a novel method to detect gastric cancer. Journal of Surgical Research, April 2006; 131(2):252-255. DOI: 10.1016/j.jss.2005.11.567

Telomerase is a ribonucleoprotein polymerase that is essential for cell immortality. Recent studies have demonstrated that a high percentage of gastric cancer tissue expressed telomerase. This study describes the presence of telomerase activity in gastric lavage fluid in patients with gastric cancer. Methods. Gastric lavage fluid was collected during esophageogastroduodenoscopy in 70 patients: 25 with gastric cancer, 25 with peptic ulcer disease, and 20 with normal stomach. The fluid and biopsy samples were analyzed for telomerase activity by a polymerase chain reaction-based telomerase repeat amplification protocol. The findings were related to the histological results. Results. Telomerase activity was present in 24 of the 25 (96%) gastric cancer tissue and in 7 of the 25 tissue specimens from peptic ulcer or gastritis. In the gastric lavage fluid, telomerase was detected in 20 patients (80%) with gastric cancer, 7 patients (28%) with peptic ulcer, and none in normal subjects (P < 0.001). The sensitivity, specificity, positive predictive value, and negative predictive value of gastric fluid telomerase expression in gastric cancer patients was 80%, 84%, 74%, and 88%, respectively. Conclusions. The presence of telomerase activity is present in gastric lavage fluid of patients with gastric cancer as compared to those without, may represent a novel method for diagnosis of gastric cancer.

C1. 68. Zhu, J., Zhang, B., Yan, X., Lao, X., and Yu, H. (2006) Preparation and characterization of chitosan/terpolymer microcapsule. [In Chinese]. Huagong Xuebao/ Journal of Chemical Industry and Engineering (China), January 2006; 57(1): 85-90.

Half N-acetylated chitosan (HNC)-based microcapsules were prepared under neutral conditions by coacervating with methacrylic acid (MAA) (20.4%)-hydroxyethyl methacrylate (HEMA) (27.4%)-methyl methacrylate (MMA) (52.2%) terpolymer (MAA-HEMA-MMA). The formed microcapsules consisted of a liquid inner core of HNC and an outer complexation shell derived from HNC and terpolymer. Prepared from 15.3% N-acetylated chitosans (FNC), HNC maintained good solubility and supplied enough protonated amino groups to coacervate with terpolymer to form microcapsules under physiological conditions. The viscosity of HNC solution between 80 and 3000 cPa·s guaranteed the formed microcapsules with good spherical shape. The preparation parameters, including pH, HNC molar mass, chitosan concentration and reaction time, greatly influenced the intrinsic coating membrane features such as morphology, thickness and network porosity, and further determined microcapsules’ permeability and mechanical stability. Low concentration of HNC favored the permeability of microcapsules, whereas high concentration was good for the resistance of microcapsules to shear force.

47. Lin, P.C., Cheng, P.C., and Yu, H. (2005) An engineered microenvironment for multi-dimensional microscopy of live cells. Scanning, November-December 2005; 27(6):284-292. DOI: 10.1002/sca.4950270603

Multidimensional imaging (MD) of live cells is gaining importance in biomedical research as the commercial availability of confocal, nonlinear optical microscopes, environmental chambers, and specific fluorescence probes grows. One crucial aspect of the MD live cell imaging involves the proper immobilization of cells, which refers to the rapid and sufficient immobilization of cells on the microscope stage, neither disrupting the cellular structure and functions nor affecting the optical properties of the cells and the environments. Conventional cell immobilization methods glue the anchoring cells to coated surfaces, but such methods require centrifugation or extended incubation and are not suitable for cells in suspension. Most of the current three-dimensional (3-D) gels either exhibit unsatisfactory optical properties or have adverse effects on cell functions in culture. Recently, an engineered 3-D microcapsule has been developed that involves the complex coacervation of a positively charged collagen and a negatively charged polymer of 2-hydroxyethyl methacrylate - methacrylic acid - methyl methacrylate (HEMA-MMA-MAA). Hence, confocal imaging of live cells in this engineered 3-D microenvironment was investigated for its optical properties and cellular function compatibility. We report here that this microenvironment facilitates efficient cell immobilization, exhibits good optical properties, and can preserve cellular structures and functions, which will be useful in MD imaging of live cells for various applications.

46. Lu, H.F., Lim, W.S., Zhang, P.C., Chia, S.M., Yu, H., Mao, H.Q., and Leong, K.W. (2005) Galactosylated Poly(vinylidene difluoride) hollow fiber bioreactor for hepatocyte culture. Tissue Engineering, November 2005; 11(11-12):1667-1677. DOI: 10.1089/ten.2005.11.1667

To overcome the limitations of long-term expression of highly differentiated hepatocyte functions, we developed a novel bioreactor in which hepatocytes were seeded in a ligand-immobilized hollow fiber cartridge. Galactosylated Pluronic polymer was immobilized on PVDF hollow fiber surface through an adsorption scheme yielding a substrate with hepatocyte-specific ligand and a hydrophilic surface layer, which could resist non-specific protein-adsorption and facilitate cell binding to the galactose ligand. Interestingly, the galactosylated PVDF hollow fiber showed enhanced serum albumin diffusion across the membrane. Freshly isolated rat hepatocytes were seeded and cultured in the extraluminal space of the hollow fiber cartridge for 18 days in a continuously circulated system. Albumin secretion function of the seeded hepatocytes was monitored by analyzing circulating medium using ELISA. The urea synthesis and P450 function (7-ethoxycoumarin dealkylase activity) were measured periodically by doping the circulating medium with NH4Cl and 7-ethoxycoumarin, respectively. Hepatocytes cultured on galactosylated PVDF hollow fibers maintained better albumin secretion and P450 functions than that on unmodified and serum-coated PVDF hollow fibers when cultured in serum-containing medium. Morphological examination by scanning electron microscopy showed that hepatocytes cultured on galactosylated PVDF hollow fibers developed significant aggregation, in contrast to those cultured on unmodified PVDF fibers or serum-coated PVDF fibers. TEM images revealed that tight junctions and canaliculus-like structures formed in these aggregates. These results suggest the potential application of this galactosylated PVDF hollow fiber cartridge for the design of bioartificial liver assist device.

45. Chia, S.M., Lin, P.C., Quek, C.H., Yin, C., Mao, H.Q., Leong, K.W., Xu, X., Goh, C.H., Ng, M.L., and Yu, H. (2005) Engineering microenvironment for expansion of sensitive anchorage-dependent mammalian cells. Journal of Biotechnology, 10 September 2005; 118(4):434-447. DOI: 10.1016/j.jbiotec.2005.05.012

Tissue engineering involves ex vivo seeding of anchorage-dependent mammalian cells onto scaffolds, or transplanting cells in vivo. The cell expansion currently requires repeated cell detachment from solid substrata by enzymatic, chemical or mechanical means. The report here presents a high yield three-dimensional culture and harvest system circumventing the conventional detachment requirements. Cells mixed with dilute cationic collagen were microencapsulated within an ultra-thin shell of synthetic polymers. The cationic collagen could rapidly form a conformal layer of collagen fibers around cells to support cell proliferation and functions. The collagen could be readily removed from cells with a buffer rinse after harvesting from the fragile microcapsules. The cells harvested from this system demonstrate improved attachment, morphology and functions over conventionally cultured cells, upon binding to ligand-conjugated polymer surfaces. The harvested cells can be re-encapsulated and allowed to proliferate again, or used immediately in applications.

44. Heng, B.C., Yu, H., Yin, Y., Lim, S.G., and Cao, T. (2005) Factors influencing stem cell differentiation into the hepatic lineage in vitro. Journal of Gastroenterology and Hepatology, July 2005; 20(7):957-987. DOI: 10.1111/j.1440-1746.2005.03856.x

A major area of research in transplantation medicine is the potential application of stem cells in liver regeneration. This would require well-defined and efficient protocols for directing the differentiation of stem cells into the hepatic lineage, followed by their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying hepatogenesis and liver metabolism. The development of pharmokinetic and cytotoxicity/genotoxicity screening tests for newly developed biomaterials and drugs, could also utilize protocols developed for the hepatic differentiation of stem cells. Hence, this review critically examines the various strategies that could be employed to direct the differentiation of stem cells into the hepatic lineage in vitro.

43. Toh, Y.C., Ho, S.T., Zhou, Y., Hutmacher, D.W., and Yu, H. (2005) Application of a polyelectrolyte complex coacervation method to improve seeding efficiency of bone marrow stromal cells in a 3D culture system. Biomaterials, July 2005; 26(19):4149-4160. DOI: 10.1016/j.biomaterials.2004.10.033

High seeding efficiency with homogenous distribution of limited cell sources such as bone marrow stromal cells (BMSCs) are of clinical relevance in scaffold-based tissue engineering. Therefore, considerable research efforts have been invested to ameliorate the seeding efficiency in 3D scaffolds. Preliminary data demonstrated that indeed BMSCs were viable and were able to proliferate in a model 3D scaffold, i.e. Cytomatrixs scaffold. However, the eventual practical application of BMSCs in such 3D scaffolds is limited by the low seeding efficiency of the cells within the scaffold. Here, we demonstrated that the cell seeding efficiency of BMSCs in the Cytomatrixs scaffold can be improved significantly (t-test, po0:05) by means of macroencapsulating the scaffold via the complex coacervation of a methylated collagen and terpolymer. The thickness and density of the polyeletrolyte complex can be modulated by the contact time between the methylated collagen and terpolymer to balance between cell entrapment efficacy and mass transfer impedance imparted by the complex. Porcine BMSCs were macroencapsulated in Cytomatrixs scaffolds using various polyelectrolyte contact time and cultured under both static and dynamic conditions. Throughout the range of contact time investigated, macroencapsulation did not affect the viability of the porcine BMSCs in dynamic culture. However, the viability of the cells under static cultures was compromised with longer polyelectrolyte contact time. Therefore, this proposed method of macroencapsulation enables customization to achieve enhanced seeding efficiency without mass transfer impedance for different culture configurations.

42. Zhu, J.H., Wang, X.W., Lao, X.J., Ng, S., Quek, C.H., Ho, H.T., and Yu, H. (2005) Encapsulating live cells with water-soluble chitosan in physiological conditions. Journal of Biotechnology, 29 June 2005; 117(4):355-365. DOI: 10.1016/j.jbiotec.2005.03.011

A new class of microcapsules was prepared under physiological conditions by polyelectrolyte complexation between two oppositely-charged, water-soluble polymers. The microcapsules consisted of an inner core of half N-acetylated chitosan and an outer shell of methacrylic acid (MAA) (20.4%)-hydroxyethyl methacrylate (HEMA) (27.4%)-methyl methacrylate (MMA)(52.2%) (MAA-HEMA-MMA) terpolymer. Both 400 and 150 kDa half N-acetylated chitosans maintained good water solubility and supplied enough protonated amino groups to coacervate with terpolymer at pH 7.0–7.4, in contrast to other chitosanbased microcapsules which must be prepared at pH <6.5. The viscosity of half N-acetylated chitosan solutions between 80 and 3000 cPa s allowed the formation of microcapsules with spherical shape. Molar mass, pH and concentration of half N-acetylated chitosan, and reaction time, influenced the morphology, thickness and porosity of the microcapsules. Microcapsules formed with high concentration of half N-acetylated chitosan exhibited improved mechanical stability, whereas microcapsules formed with low concentration of half N-acetylated chitosan exhibited good permeability. This 3D microenvironment has been configured to cultivate sensitive anchorage-dependent cells such as hepatocytes to maintain high level of functions.

41. Zhu, J.H., Zhang, B., Fang, W.W., Lao, X.J., and Yu, H. (2005) Characterization of amphoteric multilayered thin films by means of zeta potential measurements. Colloids and Surfaces B: Biointerfaces, 10 June 2005; 43(1):1-6. DOI: 10.1016/j.colsurfb.2005.03.010

Multilayer films of amphoteric methylated collagen were assembled on SOURCE 15S or SOURCE 15Q beads by sequential electrostatic deposition with negatively charged methylacrylic acid–hydroxyethyl methacrylate–methyl methacrylate (MAA–HEMA–MMA) terpolymer. Methylated collagen and terpolymer were deposited under conditions where they were oppositely charged to one another, thereby facilitating growth of the films through electrostatic interactions. Measurements revealed alternating positive and negative ζ- potential with the deposition of each methylated collagen and terpolymer layer, respectively. Assembly pH had a remarkable influence on ζ-potential of the assembled multilayers and the deposition of methylated collagen will be frustrated when the assembly pH is up to 9.0. In addition, ionic strength (NaCl concentration) showed an intricate effect on ζ-potential of the films of amphoteric methylated collagen.

40. Ng, S.S.S., Wu, Y.N., Zhou, Y., Toh, Y.E., Ho, Z.Z., Chia, S.M., Zhu, J.H., Mao, H.Q., and Yu, H. (2005) Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices. Biomaterials, June 2005; 26(16):3153-3163. DOI: 10.1016/j.biomaterials.2004.08.017

Hepatocytes are anchorage-dependent cells sensitive to microenvironment; the control of the physicochemical properties of the extra-cellular matrices may be useful to the maintenance of hepatocyte functions in vitro for various applications. In a microcapsulebased 3-D hepatocyte culture microenvironment, we could control the physical properties of the collagen nano-fibres by fine-tuning the complex-coacervation reaction between methylated collagen and terpolymer of hydroxylethyl methacrylate–methyl methacrylate–methylacrylic acid. The physical properties of the nano-fibres were quantitatively characterized usingback –scattering confocal microscopy to help optimize the physical support for hepatocyte functions. We further enhanced the chemical properties of the collagen nano-fibres by incorporating galactose onto collagen, which can specifically interact with the asialoglycoprotein receptor on hepatocytes. By correlating a range of collagen nano-fibres of different physicochemical properties with hepatocyte functions, we have identified a specific combination of methylated and galactosylated collagen nano-fibres optimal for maintaining hepatocyte functions in vitro. A model of how the physical and chemical supports interplay to maintain hepatocyte functions is discussed.

39. Toh, Y.C., Ng, S.S.S., Khong, Y.M., Samper, V., and Yu, H. (2005) A configurable 3D microenvironment in a micro-fluidic channel for primary hepatocytes culture. Assay and Drug Development Technologies, April 2005; 3(2):169-176. DOI: 10.1089/adt.2005.3.169

We have developed a technique for the in situ three-dimensional (3D) immobilization of primary rat hepatocytes within a localized matrix in a microfluidic channel that provides a 3D microenvironment incorporating both a configurable 3D matrix and fluid perfusion. This is based on the laminar flow complex coacervation of a pair of oppositely charged polyelectrolytes, i.e., methylated collagen and a terpolymer of HEMA-MMA-MAA. 3D collagen matrices were formed with minimal gelation times (< 8 min), were able to entrap cells under aqueous noncytotoxic conditions, and permitted culture media to be perfused in the microchannel by virtue of the spatial confinement of the 3D matrix on one side of the channel. The architecture and stability of the collagen matrix could be configured by the use of different material combinations and changes in the polyelectrolyte flow rates and retention time. Primary rat hepatocytes cultured for 24 h in the 3D matrix within the microchannel showed comparable or enhanced cytochrome P450 7-ethoxyresorufin-O-deethylation activity with static controls. The configurable 3D microenvironment in the microfluidic channel may be a potential 3D culture model of primary hepatocytes for drug testing applications.

38. Zhou, Y., Sun, T., Chan, M., Zhang, J., Han, Z., Wang, X., Toh, Y., Chen, J.P., and Yu, H. (2005) Scalable encapsulation of hepatocytes by electrostatic spraying. Journal of Biotechnology, 20 April 2005; 117(1):99-109. DOI: 10.1016/j.jbiotec.2004.11.004

Encapsulating cells by polyelectrolyte complex coacervation can be accomplished at physiological temperature and buffer conditions. One of the oppositely charged polyelectrolytes in the microcapsule core can be collagen or any other natural extracellular matrices suitable for cellular support while the other polyelectrolyte forms the ultra-thin shell to ensure efficient mass transfer. These microcapsules with ultra-thin shell are difficult to produce in large quantities due to their fragility. In this study, electrostatic spraying technique was used to achieve a scalable production of one such type of microcapsules formed by complex coacervation between the cationic methylated collagen and anionic terpolymer of hydroxylethyl methacrylate, methyl methacrylate and methylacrylic acid (HEMA–MMA–MAA). It was found that the microcapsule sizes were dependent on several important operational parameters, such as the diameter of the spraying needle, the flow rate of the hepatocytes–collagen mixture and the voltage of the electrical field. The microcapsules with diameters of 200–800 microm and a narrow size distribution (standard deviation of 5–28%) were successfully produced. The above parameters also influenced the hepatocyte viability and functions. With a practical encapsulation rate of up to 55 ml/h per orifice required in bio-artificial liver-assisted device applications, we have produced large quantities of microcapsules maintaining comparable cell viability (>87%), mechanical stability and bio-functions to the manually extruded microcapsules.

37. Chia, S.M., Lin, P.C., and Yu, H. (2005) TGFb1 regulation in hepatocyte-NIH3T3 co-culture is important for the enhanced hepatocyte function in 3D environment. Biotechnology and Bioengineering, 5 March 2005; 89(5):565-573. DOI: 10.1002/bit.20372

Co-culture of hepatocytes or hepatocyte spheroids with the supporting NIH3T3 in a 3D microcapsule formed with a hybrid natural/synthetic matrix has led to enhanced hepatocyte functions. We investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell–cell interactions. The conditioned media from the co-culture induced higher P450 cytochrome oxidase activity (indicated by EROD assay) in the microencapsulated hepatocytes than the conditioned media from the NIH3T3- or the hepatocytes-alone controls. Conditioned media from physically separated co-culture of hepatocytes- NIH3T3 by a membrane insert reduced the functional enhancement. Among the known stimulators of hepatocyte functions, TGFh1 is primarily responsible for the stimulation of hepatocyte functions in this 3D co-culture since the removal of TGFh1 by antibody depletion eliminated the functional enhancement and the reconstitution of TGFh1 restored the functional enhancement. Activation of latent TGFh1 in an extracellular environment were upregulated in co-culture with no observable increase in the TGFh1 expression at transcriptional and translational levels. Our data led to an improved understanding of how co-culture enhances hepatocyte functions in vitro and pave the way for further innovations in liver tissue engineering, drug metabolism studies, and other applications that require functional hepatocytes cultured in vitro.

36. Kuleshova, L.L., Ono, A., Wang, X.W., Yu, H. (2004) Current progress in vitrification of tissue-engineered constructs. Cryobiology, 49: 291-350. DOI: 10.1016/j.cryobiol.2004.10.001

Human oocytes are unique cells with regard to cryopreservation. Despite their identical morphological appearance, relevant parameters of human oocytes at the same stage of maturation vary between different infertility patients, unlike the corresponding parameters for reproductive cells of animals of the same species. This may be the critical factor responsible for the successful vitrification of mammalian oocytes—except possibly porcine oocytes—for a number of years. We suggest an approach to vitrification which enables instant regulation of the extent of penetration and dehydration of oocytes during exposure/dilution of cryoprotectant by observing the responses of the oocytes and adjusting the duration of each equilibration/dilution step to meet the individual variation in oocyte quality. Cryopreservation of immature oocytes at the GV stage seems advantageous to that of mature oocytes because these cells do not possess the sensitive, complex subcellular structure of mature oocytes; these are easily damaged by temperature reduction as previously established. Vitrification eliminates this problem as there is no need for a gradual decrease in temperature. After the first publication by Kuleshova and co-workers in 1999 (Kuleshova et al., Hum. Reprod. 14, 1999 3077–3079), several reports of live-births following vitrification of human oocytes have been published. Recent studies statistically support the advantage of vitrification over slow-cooling as a method for oocyte cryopreservation (Yoon et al., Fertil. Steril. 79, 2003 1323–1326), showing that the delivery rate for vitrified oocytes is 2.6 times better than for oocytes cryopreserved by slowcooling. There are no comparative in vivo studies on the different vitrification methods used in oocyte cryopreservation. However, we have analyzed our clinical data on the cryopreservation of 762 embryos in 267 cycles, in which different approaches and tools for cryopreservation were used. Our data displays no significant difference in pregnancy rate using either cryoloop/cryotop or straw-in-straw for vitrification of embryos. Sixteen live babies were delivered after transfer of embryos vitrified using the straw-in-straw method with a cooling rate of 400 oC/min. This has led us to believe there is no effect of different cooling/warming rates on the effectiveness of vitrification of oocytes and embryos (Kuleshova and Lopata, Fertil. Steril. 78, 2002 449–454). Cryopreservation of human oocytes is ethically advantageous and leads us forward with deeper understanding of the nature of this issue.

35. Zhang, J., Wei, H.P., Quek, C.H., Chia, S.M., and Yu, H. (2004) Quantitative measurement of collagen methylation by capillary electrophoresis. Electrophoresis, 20 October 2004; 25(20):3416-3421. DOI: 10.1002/elps.200406071

Collagen methylation has been exploited in various applications involving living cells. We have observed correlation between the collagen methylation with the rate of cell proliferation in three-dimensional (3-D) microenvironment. To quantify the degree of collagen methylation, we have developed a capillary zone electrophoresis method. Using a polyvinyl alcohol-coated fused-silica capillary and UV detection at 200 nm, we have optimized pH and separated the native collagen into three major bands in phosphate buffer (50 mM, pH 2.5) with 0.05% hydroxypropylmethylcellulose. Under these conditions, the methylated collagens were separated into four major bands, which changed with different methylation reaction conditions. We propose an index to quantify the degree of collagen methylation that also correlates with their effects on cell proliferation.

34. Santama, N., Er, C.P., Ong, L.L., and Yu, H. (2004) Distribution and functions of kinectin isoforms. Journal of Cell Sciences, 1 September 2004; 117(Pt 19):4537-4549. DOI: 10.1242/jcs.01326

Kinectin is an integral transmembrane protein on the endoplasmic reticulum, binding to kinesin, interacting with Rho GTPase and anchoring the translation elongation factor-1 complex. There has been debate on the specific role(s) of kinectin in different species and cell types. Here we identified 15 novel kinectin isoforms in the mouse nervous system, constituting a family of alternatively spliced carboxyl-terminal variants. Isoform expression is subject to cell type- and developmental stage-specific regulation. We raised specific antibodies to the kinectin variants to characterise their differential intracellular localisation and discovered that certain kinectin isoforms are found in axons where kinectin was previously believed to be absent. We also demonstrated in vivo by overexpression and RNA interference assay that kinectin is selectively involved in the transport of specific types of organelles. A 160 kDa kinectin species is mainly concentrated in the endoplasmic reticulum, anchored via its transmembrane domain and is essential for endoplasmic reticulum membrane extension. A 120 kDa kinectin species is specifically associated with mitochondria, and its interaction with kinesin was found to influence mitochondrial dynamics. These findings contribute to a more unified view of kinectin function. They suggest that different cellular processes use specific kinectin isoforms to mediate intracellular motility and targeting by transient interaction with different motor proteins or other binding partners.

33. Quek, C.H., Li, J., Sun, T., Chan, M.L., Mao, H.Q., Gan, L.M., Leong, K.W., and Yu, H. (2004) Photo-crosslinkable microcapsules formed by polyelectrolyte copolymer and modified collagen for rat hepatocyte encapsulation. Biomaterials, 17 August 2004; 25(17):3531-3540. DOI: 10.1016/j.biomaterials.2003.09.112

New anionic polyelectrolyte tetra-copolymers with photo-crosslinkable 4-(4 methoxycinnamoyl)phenyl methacrylate monomer in addition to a HEMA-MMA-MAA ter-copolymer system were synthesized. The tetra-copolymers were used to form photocrosslinkable microcapsules with modified collagen by complex coacervation for rat hepatocytes encapsulation. The hepatocytes were encapsulated within a two-layered membrane comprising of modified collagen as the inner core and an outer photocrosslinkable copolymer shell. Upon photo crosslinking of the microcapsules with UV–Vis light irradiation, the mechanical strength and chemical stability of the microcapsules, and the cellular functions of the encapsulated hepatocytes were enhanced. Particularly, the mechanical stability of the microcapsules was dramatically strengthened. The new photo-crosslinkable tetra-copolymer formulation described in this article has opened a way to the development of hepatocyte microencapsulation technology for bioartifical liver assist device.

32. Kuleshova, L.L., Wang, X.W., Wu, Y.N., Zhou, Y., and Yu, H. (2004) Vitrification of encapsulated hepatocytes with reduced cooling and warming rates. CryoLetters, July-August 2004; 25(4):241-254.

We have used microencapsulated hepatocytes as model to develop a method of vitreous cryopreservation of large quantities of cell-containing constructs. The method included a pre-equilibration procedure in which the amount of penetrating cryoprotectant was gradually increased by 15% in each step. The optimal vitrification solution consists of 40% ethylene glycol and 0.6M sucrose. The concentration of 1M sucrose used for the first dilution solution with subsequent decrease of sucrose concentration to 0.7 M sucrose and by 0.2-0.15M for each subsequent step. This sucrose dilution procedure had no adverse effect on cell functions. Three cooling rates (400°C/min and above) and three warming rates (650°C/min and above), in combination with the proposed vitrification solution, were equally effective. The optimization of the procedure and solutions allow microencapsulated hepatocytes to be preserved with almost 100% retention of cell functions and no detectable damage to the fragile microcapsules. The de-linking of the cooling/warming rates with the effectiveness of vitrification potentially paves the way for large scale cryopreservation of complex tissue engineered constructs.

31. Wang, J., Lee, I.L., Lim, W.S., Chia, S.M., Yu, H., Leong, K.W., and Mao, H.Q. (2004) Evaluation of collagen and methylated collagen as gene carriers. International Journal of Pharmaceutics, 26 July 2004; 279(1-2):115-126. DOI: 10.1016/j.ijpharm.2004.04.014

This study explores the potential of DNA complexes prepared with methylated collagen (MC) and unmodified native collagen (NC) to deliver genes into cells. The physicochemical properties and transfection abilities of these two types of complexes are studied in parallel. MC was prepared by methylation of the carboxyl groups of collagen, rendering the collagen net positively charged at neutral pH. NC/DNA complexes were prepared at pH approximately 3, but aggregated rapidly at neutral pH. These complexes did not confer significant protection to DNA due to its poor stability in serum. MC carried a positive charge at neutral pH and formed complexes with DNA in PBS; therefore MC improved DNA binding ability and the stability of the complexes at physiological conditions. MC/DNA complexes were smaller and more stable than NC/DNA complexes in PBS, and sustained released of DNA from MC/DNA complexes was observed for up to 3 weeks in PBS at 37 degrees C. In contrast, NC/DNA complexes released almost all the DNA within 6 h under the same condition. In vitro gene transfection experiments revealed that MC mediated a higher gene expression than NC, although the level of gene expression was still much lower than that achieved with polyethyleneimine/DNA complexes. In contrast to in vitro results, NC/DNA complexes yielded a 3.8-fold higher gene expression than naked DNA and MC/DNA complexes (P < 0.05) at week 2 following intramuscular injection at a DNA dose of 3microg per muscle and a weight ratio of 1. Higher weight ratios resulted in significant decrease of transfection efficiency, particularly for MC/DNA complexes. The results suggested that gene delivery via the intramuscular route followed a different mechanism that demands a different set of physiochemical properties of the carrier from other parental routes. The potential of these collagen-based gene carriers for other administration routes remain to be further investigated.

30. Sun, T., Chan, M.L., Quek, C.H., and Yu, H. (2004) Improving mechanical stability and density distribution of hepatocyte microcapsules by fibrin clot and gold nano-particles. Journal of Biotechnology, 15 July 2004; 111(2):169-177. DOI: 10.1016/j.jbiotec.2004.02.018

Bio-artificial livers (BAL) with microencapsulated hepatocytes have the typical limitations in maintaining hepatocyte functions, mechanical stability and uniform perfusion in packed or fluidized-bed bioreactors.We have previously developed microcapsules with enhanced hepatocyte functions. Here we have introduced a fibrin network inside microcapsules by (1) mixing collagen and fibrinogen with the encapsulated hepatocytes to support the cells; (2) submerging the microcapsules into a thrombin solution to induce the formation of an insoluble fibrin network inside the microcapsules. Fracture analysis on the microcapsules revealed significant improvement in mechanical stability. We have also introduced different amounts of gold nano-particles into microcapsules to achieve different densities for uniform bioreactor perfusion. These gold nano-particles also improved the mechanical stability of the microcapsules. Both the fibrin network and gold nano-particles exhibited the additional benefits of enhancing certain bio-functions of the encapsulated hepatocytes. The applications of these improved microcapsules in the development of bio-artificial livers are discussed.

29. Ng, S.C., Heng, B.C., and Yu, H. (2004) Slow-cooling protocols for microcapsule cryopreservation. Journal of Microencapsulation, June 2004; 21(4):455-467. DOI: 10.1080/02652040410001729250

The relatively large size (300–400 mm) and fragile semi-permeable membrane of microcapsules makes them particularly prone to cryodamage. This study investigated slow-cooling protocols for the cryopreservation of microcapsules. Instead of a programmable freezing-machine, slow cooling was carried out directly within a -80 degrees C refrigerator. A range of increasing cryoprotectant (DMSO and EG) concentrations with slow cooling was investigated. The results showed that 2.8M (20% v/v) DMSO and 2.7M (15% v/v) EG were optimal for microcapsule cryopreservation, resulting in approximately 55–60% of the microcapsules remaining intact, with a relatively high post-thaw cell viability of 80–85%. Post-thaw cell viability and microcapsule integrity were consistently higher at equivalent molarities of DMSO compared to EG. Hence, all subsequent studies utilized only DMSO. Post-thaw cell viability upon slow cooling with 2.8 M (20% v/v) DMSO was significantly improved in the presence of 0.25M sucrose (>95%), but there was no enhancement in microcapsule integrity. Neither post-thaw cell viability nor microcapsule integrity was improved with multi-step exposure and removal of sucrose, compared to a single-step protocol. There was also no improvement in either post-thaw cell viability or microcapsule integrity in the presence of 20% (w/v) Ficoll. Hence, the optimal condition for microcapsule cryopreservation by slow-cooling is with 2.8M (20% v/v) DMSO and 0.25M sucrose.

28. Heng, B.C., Yu, H., and Ng, S.C. (2004) Strategies for the cryopreservation of microencapsulated cells. Biotechnology and Bioengineering, 20 January 2004; 85(2):202-213. DOI: 10.1002/bit.10837

The major challenge in developing cryopreservation protocols for microencapsulated cells is that the relatively large size (300–400 Am) and the fragile semipermeable membrane of microcapsules makes them particularly prone to cryodamage. Rapid-cooling cryopreservation protocols with high DMSO concentrations (3.5M, 25% v/v) resulted in low post-thaw cell viability (<10%), which did not improve with higher concentrations (4.5M, 32% v/v) and longer exposure to DMSO, even though the majority of microcapsules (60–80%) remained intact. Subsequent investigations of slow cooling with a range of DMSO and EG concentrations resulted in a much higher post-thaw cell viability (80–85%), with the majority of the microcapsules remaining intact (approximately 60%) when DMSO was used at a concentration of 2.8M (20% v/v) and EG at a concentration of 2.7M (15% v/v). The presence of 0.25M sucrose significantly improved post-thaw cell viability upon slow cooling with 2.8M (20% v/v) DMSO, although it had no effect on microcapsule integrity. Multistep exposure and removal of sucrose did not significantly improve either postthaw cell viability or microcapsule integrity, compared to a single-step protocol. Ficoll 20% (w/v) also did not significantly improve post-thaw cell viability and microcapsule integrity. Hence, the optimal condition for microcapsule cryopreservation developed in this study is slow cooling with 2.8M (20% v/v) DMSO and 0.25M sucrose.

27. Li, Y., Wang, J., Lee, C., Wang, C.Y., Gao, S.J., Tong, G.P., Ma, Y.X., Yu, H., Mao, H.Q., Leong, K.W., and Wang, S. (2004) CNS gene transfer facilitated by a novel controlled release system based on DNA complexes of degradable polycation PPE-EA: A comparison with Polyethylenimine/DNA complexes. Gene Therapy, 11(1):109-114. DOI: 10.1038/sj.gt.3302135

Nonviral gene delivery systems based upon polycation/plasmid DNA complexes are quickly gaining recognition as an alternative to viral gene vectors for their potential in avoiding immunogenicity and toxicity problems inherent in viral systems. We investigated in this study the feasibility of using a controlled release system based on DNA complexed with a recently developed polymeric gene carrier, polyaminoethyl propylene phosphate (PPE-EA), to achieve gene transfer in the brain. A unique feature of this gene delivery system is the biodegradability of PPE-EA, which can provide a sustained release of DNA at different rates depending on the charge ratio of the polymer to DNA. PPE-EA/DNA complexes, naked DNA, and DNA complexed with polyethylenimine (PEI), a nondegradable cationic polymer known to be an effective gene carrier, were injected intracisternally into the mouse cerebrospinal fluid. Transgene expression mediated by naked DNA was mainly detected in the brain stem, a region close to the injection site. With either PPE-EA or PEI as a carrier, higher levels of gene expression could be detected in the cerebral cortex, basal ganglia, and diencephalons. Transgene expression in the brain mediated by PPE-EA/DNA complexes at an N/P ratio of 2 persisted for at least 4 weeks, with a significant higher level than that produced by either naked plasmid DNA or PEI/DNA at the 4-week time point. Furthermore, PPE-EA displayed much lower toxicity in cultured neural cells as compared to PEI and did not cause detectable pathological changes in the central nervous system (CNS). The results established the potential of PPE-EA as a new and biocompatible gene carrier to achieve sustained gene expression in the CNS.

26. Lu, H.F., Lim, W.S., Wang, J., Tang, Z.Q., Zhang, P.C., Leong, K.W., Chia, S.M., Yu, H., and Mao, H.Q. (2003) Galactosylated PVDF membrane promotes hepatocyte attachment and functional maintenance. Biomaterials, December 2003; 24(27):4893-4903. DOI: 10.1016/S0142-9612(03)00404-6

One of the major challenges in BLAD design is to develop functional substrates suitable for hepatocyte attachment and functional maintenance. In the present study, we designed a poly(vinylidene difluoride) (PVDF) surface coated with galactose-tethered Pluronic polymer. The galactose-derived Pluronic F68 (F68-Gal) was adsorbed on PVDF membrane through hydrophobic–hydrophobic interaction between PVDF and the polypropylene oxide segment in Pluronic. The galactose density on the modified PVDF surface increased with the concentration ofthe F68-Gal solution, reaching 15.4 nmol galactosyl groups per cm2 when a 1 mg/ml ofF68-Gal solution was used. The adsorbed F68-Gal remained relatively stable in culture medium. Rat hepatocytes attachment efficiency on F68-Gal modified PVDF membrane was similar to that on collagen-coated surface. The attached hepatocytes on PVDF/F68-Gal membrane self-assembled into multi-cellular spheroids after 1 day of culture. These attached hepatocytes in spheroids exhibited higher cell functions such as albumin synthesis and P450 1A1 detoxification function compared to unmodified PVDF membrane and collagen-coated surface. These results suggest the potential of this galactose-immobilized PVDF membrane as a suitable substrate for hepatocyte culture.

25. Wong, S.C.H., Ong, L.L., Er, C.P., Gao, S., Yu, H., and So, J.B. (2003) Cloning of rat telomerase catalytic subunit functional domains, reconstitution of telomerase activity and enzymatic profile of pig and chicken tissues. Life Sciences, 10 October 2003; 73( 21):2749-2760. DOI: 10.1016/S0024-3205(03)00670-2

Telomerase is a ribonucleoprotein polymerase which adds TTAGGG repeats to telomeric ends. Recent studies reported the reverse transcription enzyme activity mostly from the catalytic subunit (TERT) of the enzyme complex. Both human telomerase catalytic subunit (hTERT) and mouse telomerase catalytic subunit (mTERT) had been previously cloned but not rat telomerase catalytic subunit rTERT. In this study, the rTERT functional domains were cloned and was found that its function resemble to mouse and human telomerase. In addition, chicken and pig telomerase activity profile were studied and its enzyme activity is related to its proliferation capability of individual tissues. However, its catalytic subunit does not like mouse, rat and human cases that the telomerase activity could not reconstituted by the in-vitro transfection of mTERT and hTERT cloned vectors. Here we demonstrated that rTERT is similar to mTERT and hTERT but not pig and chicken telomerase. Further studies are needed to verify the malignancy characteristics because nowadays artificial organs/tissues from these animals are used for the transplantation to human body.

24. Sun, T., Chan, M.L., Zhou, Y., Xu, X., Zhang, J., Lao, X., Wang, X., Quek, C.H., Chen, J.P., Leong, K.W., and Yu, H. (2003) Use of ultrathin shell microcapsules of hepatocytes in bioartificial liver-assist device. Tissue Engineering, 30 August 2003; 9(supplement 1):65-75. DOI: 10.1089/10763270360696987

We previously encapsulated hepatocytes in ultrathin shell microcapsules and showed them to have enhanced differentiated functions over cells cultured in monolayer. Here we have used these microencapsulated hepatocytes in a bioartificial liver-assisted device (BLAD) with a rat hepatectomy model. Primary rat hepatocytes were encapsulated in 150- to 200-m microcapsules, using an electrostatic droplet generator. The microencapsulated hepatocytes exhibited good in vitro urea synthesis activity in plasma from rats with fulminant hepatic failure (FHF). The ex vivo hemoperfusion was conducted in FHF rats by perfusing plasma at a rate of 1–2 mL/min through 1.5–2 108 encapsulated hepatocytes packed into a packed-bed bioreactor. Hemoperfusion with the bioreactor was initiated 5 h after operative induction of liver failure and continued for 7 h. The BLAD-treated rats showed improvements over the control groups in survival time and metabolic indicators, including ammonia and total bilirubin levels. Furthermore, expanded bed adsorption (EBA) detoxification technology using Streamline-SP resin was explored to complement the bioreactor with microencapsulated hepatocytes. In vitro experiments indicated that serum ammonia could be specifically removed in dose-dependent manner, whereas the total serum proteins were unaffected by the resin. In ex vivo experiments, hemoperfusion with the resin was initiated 3 h after operative induction of liver failure and continued for 7 h. The resin-treated rats showed obvious serum ammonia removal with no observable total blood protein and blood cell adsorption. Therefore, Streamline-SP resin can potentially be integrated into a BLAD for improved efficacy.

23. Ong, L.L., Er, C.P., Ho, A., Aung, M.T., and Yu, H. (2003) Kinectin anchors the translation elongation factor-1Delta to the endoplasmic reticulum. Journal of Biological Chemistry, 22 August 2003; 278(34):32115-32123. DOI: 10.1074/jbc.M210917200

Kinectin has been proposed to be a membrane anchor for kinesin on intracellular organelles. A kinectin isoform that lacks a major portion of the kinesin-binding domain does not bind kinesin but interacts with another resident of the endoplasmic reticulum, the translation elongation factor-1 delta (EF-1δ). This was shown by yeast two-hybrid analysis and a number of in vitro and in vivo assays. EF-1δ provides the guanine nucleotide exchange activities on EF-1δ during elongation step of protein synthesis. The minimal EF-1_-binding domain on kinectin resides within a conserved region present in all the kinectin isoforms. Overexpression of the kinectin fragments in vivo disrupted the intracellular localization of EF-1δ proteins. This report provides evidence of an alternative kinectin function as the membrane anchor for EF-1δ on the endoplasmic reticulum and provides clues to the EF-1 complex assembly and anchorage on the endoplasmic reticulum.

22. Xu, X., Yee, W.C., Hwang, P.Y., Yu, H., Wan, A.C., Gao, S., Boon, K.L., Mao, H.Q., Leong, K.W., and Wang, S. (2003) Peripheral nerve regeneration with sustained release of poly(phosphoester) microencapsulated nerve growth factor within nerve guide conduits. Biomaterials, June 2003; 24(13):2405-2412. DOI: 10.1016/S0142-9612(03)00109-1

Prolonged delivery of neurotrophic proteins to the target tissue is valuable in the treatment of various disorders of the nervous system. We have tested in this study whether sustained release of nerve growth factor (NGF) within nerve guide conduits (NGCs), a device used to repair injured nerves, would augment peripheral nerve regeneration. NGF-containing polymeric microspheres fabricated from a biodegradable poly(phosphoester) (PPE) polymer were loaded into silicone or PPE conduits to provide for prolonged, site-specific delivery of NGF. The conduits were used to bridge a 10mm gap in a rat sciatic nerve model. Three months after implantation, morphological analysis revealed higher values of fiber diameter, fiber population and fiber density and lower Gratio at the distal end of regenerated nerve cables collected from NGF microsphere-loaded silicone conduits, as compared with those from control conduits loaded with either saline alone, BSA microspheres, or NGF protein without microencapsulation. Beneficial effects on fiber diameter, G-ratio and fiber density were also observed in the permeable PPE NGCs. Thus, the results confirm a long term promoting effect of exogenous NGF on morphological regeneration of peripheral nerves. The tissue-engineering approach reported in this study of incorporation of a microsphere protein release system into NGCs holds potential for improved functional recovery in patients whose injured nerves are reconstructed by entubulation.

21. Yin, C., Chia, S.M., Quek, C.H., Yu, H., Zhuo, R.X., Leong, K.W., and Mao, H.Q. (2003) Microcapsules with improved mechanical stability for hepatocyte culture. Biomaterials, May 2003; 24(10):1771-1780. DOI: 10.1016/S0142-9612(02)00580-X

Packed-bed or fluidized-bed bioreactor filled with microencapsulated hepatocytes has been proposed as one of the promising designs for bioartificial liver assist device (BLAD) because of potential advantages of high mass transport rate and optimal microenvironment for hepatocyte culture. Recently, we have developed a microcapsule system for the encapsulation of hepatocytes. The microcapsules consist of an inner core ofmodified collagen and an outer shell ofterpolymer of methyl methacrylate, methacrylate and hydroxyethyl methacrylate. Cells encapsulated in these microcapsules exhibit enhanced cellular functions. Improving the mechanical stability of the microcapsules to withstand the shear stress induced by high perfusion rate would be crucial to the success of BLAD applications. In this study, we investigated the effects of terpolymer molecular weight (Mw) on the mechanical property of these microcapsules and the differentiated functions of encapsulated hepatocytes. Six terpolymers with different Mw were synthesized using radical polymerization in solution by adjusting the reaction temperature and the initiator concentration. All the terpolymers formed microcapsules with the methylated collagen. While the terpolymer Mw had little effect on the capsule membrane thickness and permeability ofserum albumin, the mechanical property of the microcapsules was significantly improved by the higher Mw of the terpolymer. Differentiated functions of the hepatocytes cultured in the microcapsules, including urea synthesis, albumin synthesis and cytochrome P450 metabolic activity, were not significantly affected by the terpolymer Mw.

20. Wong, S.C.H., Yu, H., Moochhala, S.M., and So, J.B. (2003) Antisense telomerase induced cell growth inhibition, cell cycle arrest and telomerase activity down-regulation in gastric and colon cancer cells. Anticancer Research, January-February 2003; 23(1A):465-469.

BACKGROUND: Antisense telomerase RNA (anti-hTR) coupled with 2',5'-linked tetraadenylate (2-5A) shows an inhibitory effect on cell growth and induces apoptosis in certain kinds of cancer. This experiment examined the anti-hTR effect on gastric and colorectal cancer cells. MATERIALS AND METHODS: Gastric cancer cells CRL-5822, CRL-5971 and colorectal cancer cells HTB-38, CCL-247 were treated with repeated doses of anti-hTR. Cell growth and cell cycle parameters were analysed. Telomerase activity was measured by TRAP assay. Control oligonucleotides and normal fibroblast were used as control. RESULTS: After two doses of anti-hTR, the cell viability for CRL-5822, CRL-5971, HTB-38 and CCL-247 cells was reduced to 15%, 12%, 37% and 37%, respectively. The DNA histogram showed cells arrested at the G2/M-phase and the sub-G1 peak found indicated that apoptosis had occurred. CONCLUSION: Anti-hTR demonstrated inhibition of cell growth and cell cycle arrest on gastric and colorectal cancer cells through a telomerase regulation pathway. Its specificity towards cancerous tissues but not normal tissues suggested a potential for anti-cancer therapy.

19. Xu, X., Yu, H., Gao, S., Mao, H.Q., Leong, K.W., and Wang, S. (2002) Polyphosphoester microspheres for sustained release of biologically active nerve growth factor. Biomaterials, September 2002; 23(17):3765-3772. DOI: 10.1016/S0142-9612(02)00116-3

Controlled delivery of neurotrophic proteins to a target tissue by biodegradable polymer microspheres has been explored widely for its potential applications in the treatment of various disorders in the nervous system. We investigated in this study the potential of polyphosphoester microspheres as carriers for the sustained release of nerve growth factor (NGF), a water-soluble neurotrophic protein. Two polyphosphoesters (PPEs), P(BHET-EOP/TC) and P(DAPG-EOP), as well as poly(lactide/glycolic acid) (PLGA), were used to fabricate microspheres by a W/O/W emulsion and solvent evaporation/extraction method. With bovine serum albumin as a model protein to optimize processing parameters, P(DAPG-EOP) microspheres exhibited a lower burst effect but similar protein entrapment levels and efficiencies when compared with those made of PLGA. Bioactive NGF could be released for at least 10 weeks from the P(DAPG-EOP) microspheres, as confirmed by a neurite outgrowth assay of the PC12 cells. These NGF containing microspheres were incorporated into the nerve guide conduits that were implanted to bridge a 10mm gap in a rat sciatic nerve model. Two weeks after implantation, immunostaining with an antibody against the neurofilament protein confirmed the presence of axons at the distal end of regenerated cables within the NGF microsphere-loaded conduits. These results demonstrated the feasibility of using biodegradable PPEs for microencapsulation of NGF and provided a basis for future therapeutic application of the microspheres.

18. Chia, S.M., Wan, A.C., Quek, C.H., Mao, H.Q., Xu, X., Lu, S., Ng, M.L., Leong, K.W., and Yu, H. (2002) Multi-layered microcapsules for cell encapsulation. Biomaterials, February 2002; 23(3):849-856. DOI: 10.1016/S0142-9612(01)00191-0

Mechanical stability, complete encapsulation, selective permeability, and suitable extra-cellular microenvironment, are the major considerations in designing microcapsules for cell encapsulation. We have developed four types of multi-layered microcapsules that allow selective optimization of these parameters.Primary hepatocytes were used as model cells to test these different microcapsule configurations.Type-I microcapsules with an average diameter of 400 mm were formed by complexing modified collagen with a terpolymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2–5 mm.Cells in these microcapsules exhibited improved cellular functions over those cultured on collagen monolayers.Type-II microcapsules were formed by encapsulating the Type-I microcapsules in another 2–5 mm ter-polymer shell and a B5 mm collagen layer between the two ter-polymer shells to ensure complete cell encapsulation.Type-III microcapsules comprised of a macro-porous exoskeleton with materials such as alumina sol–gel coated on the Type-I microcapsules.Nano-indendation assay indicated an improved mechanical stability over the Type-I microcapsules.Type-IV microcapsules were created by encapsulating Type-III microcapsules in another 2–5 mm ter-polymer shell, with the aim of imparting a negatively charged smooth surface to minimize plasma protein absorption and ensure complete cell encapsulation. The permeability for nutrient exchange, cellular functions in terms of urea production and mechanical stability of the microcapsules were characterized. The advantages and limitations of these microcapsules for tissue engineering are discussed.

17. Al-Haddad, A., Shonn, M.A., Redlich, B., Blocker, A., Burkhardt, J.K., Yu, H., Hammer, J.A., Weiss, D.G., Steffen, W., Griffiths, G., and Kuznetsov, S.A. (2001) Myosin Va bound to phagosomes binds to F-Actin and delays microtubule-dependent motility. Molecular Biology of the Cell, September 2001; 12(9):2742-2755.

We established a light microscopy-based assay that reconstitutes the binding of phagosomes purified from mouse macrophages to preassembled F-actin in vitro. Both endogenous myosin Va from mouse macrophages and exogenous myosin Va from chicken brain stimulated the phagosome–F-actin interaction. Myosin Va association with phagosomes correlated with their ability to bind F-actin in an ATP-regulated manner and antibodies to myosin Va specifically blocked the ATP-sensitive phagosome binding to F-actin. The uptake and retrograde transport of phagosomes from the periphery to the center of cells in bone marrow macrophages was observed in both normal mice and mice homozygous for the dilute-lethal spontaneous mutation (myosin Va null). However, in dilute-lethal macrophages the accumulation of phagosomes in the perinuclear region occurred twofold faster than in normal macrophages. Motion analysis revealed saltatory phagosome movement with temporarily reversed direction in normal macrophages, whereas almost no reversals in direction were observed in dilute-lethal macrophages. These observations demonstrate that myosin Va mediates phagosome binding to F-actin, resulting in a delay in microtubuledependent retrograde phagosome movement toward the cell center. We propose an “antagonistic/cooperative mechanism” to explain the saltatory phagosome movement toward the cell center in normal macrophages.

16. Wang, S., Ma, N., Gao, S.J., Yu, H., and Leong, K.W. (2001) Transgene expression in the brainstem affected by intramuscular injection of Polyethylenimine/DNA complexes. Molecular Therapy, May 2001; 3(5pt1):658-664. DOI: 10.1006/mthe.2001.0324

Gene delivery into the CNS without tissue destruction is challenging. As neurons are capable of taking up exogenous particulates from the muscles that they innervate, we investigated the feasibility of achieving gene transfer in CNS neurons by peripheral intramuscular injection of plasmid DNA complexed with the cationic polymer polyethylenimine (PEI) in the rat hypoglossal system. Using the luciferase reporter gene driven by a Rous sarcoma virus promoter, transgene expression of up to 4 3 106 RLU per brain stem at 20 mg of plasmid DNA was achieved after tongue injection. Using lacZ as a reporter gene, transgene expression in the brain stem was detected in hypoglossal motor neurons, a group of neurons that innervate tongue muscles. The plasmid DNA was detected by PCR analysis in the brain-stem samples, demonstrating that the PEI/DNA complexes had migrated by retrograde axonal transport to neuronal cell bodies in the brain stem after being internalized by nerve terminals in the tongue muscle. Using a therapeutic bcl-2 gene driven by a cytomegalovirus promoter and Western blotting, transgene expression was detectable in the brain stem as early as 18 h after tongue injection and lasted for at least 2 weeks. Two lipid transfection agents, GenePORTER and TransFast, mediated a weak gene expression in the hypoglossal system, but not two polymers, poly-L-lysine and chitosan. The nonviral neuronal gene delivery method established in this study bypasses the blood–brain barrier and suggests a possible therapeutic strategy for noninvasive CNS gene transfer.

15. Wan, A.C., Mao, H.Q., Wang, S., Leong, K.W., Ong, L.K., and Yu, H. (2001) Fabrication of poly(phosphoester) nerve guides by immersion precipitation and the control of porosity. Biomaterials, May 2001; 22(10):1147-1156. DOI: 10.1016/S0142-9612(00)00355-0

Immersion precipitation was employed as a method for the fabrication of polymeric conduits from P(BHET-EOP/TC), a poly(phosphoester) with an ethylene terephthalate backbone, to be applied as guidance channels for nerve regeneration. Coatings of various porosities could be obtained by immersing mandrels coated with a solution of the polymer in chloroform into non-solvent immersion baths, followed by freeze or vacuum-drying. The porosity of the coatings decreased with an increase in polymer molecular weight, drying time before precipitation and concentration of polymer solution. The effects of these parameters can be rationalized by employing ternary phase diagrams, where porosity is directly related to the degree of phase separation available to the system before gelation occurs. To afford improved porosity control, a new system was developed which employed the contrasting phase-separation behavior of P(BHET-EOP/TC)/chloroform solution in methanol and water. As water is essentially a non-solvent for the polymer, the demixing boundary of the P(BHET-EOP/TC)-CHCl3 H2O system is located close to the polymer-solvent edge of the phase diagram, while that of the P(BHET-EOP/TC)-CHCl3-MeOH system is located further away. A mixture of methanol and water allows the demixing boundary to be shifted to intermediate coordinates. By immersing P(BHET-EOP/TC) coatings in immersion baths containing different ratios of water and methanol, then gradually titrating the bath with methanol to a concentration of 70% (v/v) methanol, surface porosities ranging from 2 to 58% could be achieved.

14. Wang, S., Wan, A.C., Xu, X., Gao, S., Mao, H.Q., Leong, K.W., and Yu, H. (2001) A new nerve guide conduit material composed of a biodegradable poly(phosphoester). Biomaterials, May 2001; 22(10):1157-1169. DOI: 10.1016/S0142-9612(00)00356-2

There is a resurgence of interest in the development of degradable and biocompatible polymers for fabrication of nerve guide conduits (NGCs) in recent years. Poly(phosphoester) (PPE) polymers are among the attractive candidates in this context, in view of their high biocompatibility, adjustable biodegradability, flexibility in coupling fragile biomolecules under physiological conditions and a wide variety of physicochemical properties. The feasibility of using a biodegradable PPE, P(BHET-EOP/TC), as a novel NGC material was investigated. Two types of conduits were fabricated by using two batches of P(BHET-EOP/TC) with different weight-average molecular weights (Mw) and polydispersity indexes (PI). The polymers as well as conduits were non-toxic to all six types of cells tested, including primary neurones and neuronally differentiated PC12 cells. After in situ implantation in the sciatic nerve of the rat, two types of conduits triggered a similar tissue response, inducing the formation of a thin tissue capsule composed of approximately eight layers of "broblasts surrounding the conduits at 3 months. Biological performances of the conduits were examined in the rat sciatic nerve model with a 10mm gap. Although tube fragmentation, even tube breakage, was observed within less than 5 days post-implantation, successful regeneration through the gap occurred in both types of conduits, with four out of 10 in the Type I conduits (Mw14,900 and PI 2.57) and 11 out of 12 in the Type II conduits (Mw 18,900 and PI 1.72). The degradation of conduits was further evidenced by increased roughness on the tube surface in vivo under scanning electron microscope and a mass decrease in a time-dependent manner in vitro. The Mw of the polymers dropped 33 and 24% in the Type I and II conduits, respectively, in vitro within 3 months. Among their advantages over other biodegradable NGCs, the PPE conduits showed negligible swelling and no crystallisation after implantation. Thus, these PPE conduits can be effective aids for nerve regeneration with potential to be further developed into more sophisticated NGCs that have better control of the conduit micro-environment for improved nerve regeneration.

13. Chen, J.P., and Yu, H. (2000) Lead removal from synthetic wastewater by crystallization in a fluidized-bed reactor. Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 35(6):817-835.

A fluidized‐bed reactor (FBR) was employed in the study to remove lead from the synthetic wastewater by crystallization of metal carbonate precipitates on surfaces of the sand grains. For the influent concentration up to 40 mg/L, lead removal efficiency reached 99 % and the effluent concentration was less than 1 mg/L when the system was operated with a series of optimum conditions. Feed ratio CT/[Pb] and recycle ratio should be maintained at 3 mol/mol and 0.67, respectively, and the hydraulic load should not be more than 22 m/h. The optimum pH for lead carbonate crystallization was 8 to 9, while the ratio of bed height to total height of the FBR was 0.25 to 0.3. In addition, the stable operation in terms of lead removal and solution turbidity was observed after a 380‐minute operation. Analysis of the composition of crystals deposited on the sand grains surface showed that nearly 99 % was lead salt. Furthermore the lead ions can be easily recovered by adding hydrochloride acid. This lead solution could be suitable for further industrial purposes.

12. Ong, L.L., Lim, A.P., Er, C.P., Kuznetsov, S.A., and Yu, H. (2000) Kinectin-Kinesin binding domains and their effects on organelle motility. Journal of Biological Chemistry, 20 October 2000; 275(42):32854-32860. DOI: 10.1074/jbc.M005650200

Intracellular organelle motility involves motor proteins that move along microtubules or actin filaments. One of these motor proteins, kinesin, was proposed to bind to kinectin on membrane organelles during movement. Whether kinectin is the kinesin receptor on organelles with a role in organelle motility has been controversial. We have characterized the sites of interaction between human kinectin and conventional kinesin using in vivo and in vitro assays. The kinectin-binding domain on the kinesin tail partially overlaps its head-binding domain and the myosin-Va binding domain. The kinesin-binding domain on kinectin resides near the COOH terminus and enhances the microtubulestimulated kinesin-ATPase activity, and the overexpression of the kinectin-kinesin binding domains inhibited kinesin-dependent organelle motility in vivo. These data, when combined with other studies, suggest a role for kinectin in organelle motility.

11. Chia, S.M., Leong, K.W., Li, J., Xu, X., Zeng, K., Er, P.N., Gao, S., and Yu, H. (2000) Hepatocyte encapsulation for enhanced cellular functions. Tissue Engineering, October 2000; 6(5):481-495. DOI: 10.1089/107632700750022134

An efficient bioartificial liver-assisted device can sustain the lives of patients with acute liver failure. Among different configurations of the bioreactor design, hepatocyte encapsulation has important features that satisfy most requirements of the device. We have encapsulated rat hepatocytes in a two-layer polymeric membrane by complex coacervation using a simple setup and demonstrated enhanced cellular functions up to three times higher than those of the monolayer control. These microcapsules of the functioning hepatocytes have a 2- to 3-mm outer layer of synthetic polymer with 25% 2-hydroxyethyl methacrylate, 25% methacrylic acid, and 50% methyl methacrylate and an inner layer of positively charged modified collagen as a suitable substrate for the enhanced cellular functions. Permeable only to small molecules up to albumin, the microcapsules should allow unimpeded exchange of nutrients, oxygen, growth factors, and metabolites but prevent attack by immunoglobulins of the immune system, and no “skin effect” of the collagen has been observed. Mechanical properties of the microcapsules measured with a nano-indentation method suggest that the microcapsules should be suitable for use in a bioartificial liver-assisted device.

10. Cai, Y.D., Yu, H., and Chou, K.C. (1998) Artificial neural network method for predicting HIV protease cleavage sites in protein. Journal of Protein Chemistry, 17(7):607-615. DOI: 10.1007/BF02780962

Knowledge of the polyprotein cleavage sites by HIV protease will refine our understanding of its specificity, and the information thus acquired will be useful for designing specific and efficient HIV protease inhibitors. The search for inhibitors of HIV protease will be greatly expedited if one can find an accurate, robust, and rapid method for predicting the cleavage sites in proteins by HIV protease. In this paper, Kohonen's self-organization model, which uses typical artificial neural networks, is applied to predict the cleavability of oligopeptides by proteases with multiple and extended specificity subsites. We selected HIV-1 protease as the subject of study. We chose 299 oligopeptides for the training set, and another 63 oligopeptides for the test set. Because of its high rate of correct prediction (58/63 = 92.06%) and stronger fault-tolerant ability, the neural network method should be a useful technique for finding effective inhibitors of HIV protease, which is one of the targets in designing potential drugs against AIDS. The principle of the artificial neural network method can also be applied to analyzing the specificity of any multisubsite enzyme.

9. Cai, Y.D., Yu, H., and Chou, K.C. (1998) Prediction of beta-turns. Journal of Protein Chemistry, 17(4):363-376. DOI: 10.1023/A:1022559300504

Kohonen's self-organization model, a neural network model, is applied to predict the p-turns in proteins. There are 455 p-turn tetrapeptides and 3807 non-p-turn tetrapeptides in the training database. The rates of correct prediction for the 110 p-turn tetrapeptides and 30,229 non-p-turn tetrapeptides in the testing database are 81.8% and 90.7%, respectively. The high quality of prediction of neural network model implies that the residue-coupled effect along a polypeptide chain is important for the formation of reversal turns, such as p-turns, during the process of protein folding.

8. Rao, P.N., Yu, H., Hodge, R., Pettenati, M.J., and Sheetz, M.P. (1997) Assignment of the human kinectin gene (KTN1), encoding a kinesin-binding protein, to chromosome 14 band q22.1 by in situ hybridization. Cytogenetics Cell Genetics, 79(3-4):196-197.

Movement and reorganization of organelles in cells is brought about by a transport machinery made up of microtubules, and its associated motor proteins, kinesin, and dynein (Vallee and Sheetz, 1996). Kinectin (KTN1), is an important vesicle membrane protein that acts to bind the vesicles to kinesin, and promotes vesicle motility (Kumar et al., 1995). The human kinectin cDNA clone was isolated from an embryonic chick brain cDNA library by immune-screening with a panel of monoclonal antibodies. It encodes a 1364-amino acid protein of 156 kDa which contains an N-terminal transmembrane domain and two C-terminal leucine zipper motifs (Yu et al., 1995; Futterer et al., 1995). Print et al. (1996), isolated a full length cDNA (CG-1), from a human leukocyte library which was similar in size and structure to the KTN1 gene and was found to be expressed in diverse human tissues. They mapped CF-1 to 14q22. We report the confirmation of the regional localization of this important motor protein gene, kinectin (KTN1), by fluorescence in situ hybridization to 14q22.1.

7. Cai, Y.D., Yu, H., and Chou, K.C. (1997) Artificial neural network method for predicting the specificity of galNAc-transferase. Journal of Protein Chemistry, October 1997; 16(7):689-700. DOI: 10.1023/A:1026306520790

The specificity of GalNAc-transferase is consistent with the existence of an extended site composed of nine subsites, denoted by R4, R3, R2, R1, R0, R1, R2, R3, and R4, where the acceptor at R0, is either Ser or Thr to which the reducing monosaccharide is anchored. To predict whether a peptide will react with the enzyme to form a Ser- or Thr-conjugated glycopeptide, a neural network method—Kohonen's self-organization model is proposed in this paper. Three hundred five oligopeptides are chosen for the training site, with another 30 oligopeptides for the test set. Because of its high correct prediction rate (26/30 = 86.7%) and stronger fault-tolerant ability, it is expected that the neural network method can be used as a technique for predicting O-glycosylation and designing effective inhibitors of GalNAc-transferase. It might also be useful for targeting drugs to specific sites in the body and for enzyme replacement therapy for the treatment of genetic disorders.

6. Blocker, A., Severin, F.F., Burkhardt, J.K., Bingham, J.B., Yu, H., Olivo, J.C., Schroer, T.A., Hyman, A.A., and Griffiths, G. (1997) Molecular requirements for bi-directional movement of phagosomes along microtubules. Journal of Cell Biology, 7 April 1997; 137(1):113-129.

Microtubules facilitate the maturation of phagosomes by favoring their interactions with endocytic compartments. Here, we show that phagosomes move within cells along tracks of several microns centrifugally and centripetally in a pH- and microtubuledependent manner. Phagosome movement was reconstituted in vitro and required energy, cytosol and membrane proteins of this organelle. The activity or presence of these phagosome proteins was regulated as the organelle matured, with “late” phagosomes moving threefold more frequently than “early” ones. The majority of moving phagosomes were minus-end directed; the remainder moved towards microtubule plus-ends and a small subset moved bi-directionally. Minus-end movement showed pharmacological characteristics expected for dyneins, was inhibited by immunodepletion of cytoplasmic dynein and could be restored by addition of cytoplasmic dynein. Plus-end movement displayed pharmacological properties of kinesin, was inhibited partially by immunodepletion of kinesin and fully by addition of an anti-kinesin IgG. Immunodepletion of dynactin, a dynein-activating complex, inhibited only minus-end directed motility. Evidence is provided for a dynactin-associated kinase required for dyneinmediated vesicle transport. Movement in both directions was inhibited by peptide fragments from kinectin (a putative kinesin membrane receptor), derived from the region to which a motility-blocking antibody binds. Polypeptide subunits from these microtubule-based motility factors were detected on phagosomes by immunoblotting or immunoelectron microscopy. This is the first study using a single in vitro system that describes the roles played by kinesin, kinectin, cytoplasmic dynein, and dynactin in the microtubule-mediated movement of a purified membrane organelle.

5. Sheetz, M. P. and Yu, H. (1996) Regulation of the kinesin and cytoplasmic dynein driven organelle motility. Seminars in Cell & Developmental Biology, June 1996; 7(3):329-334.

We propose that the regulation of kinesin- and cytoplasmic dynein-driven organelle motility is coupled since membrane traffic within cells is largely cyclic and both motors are required to complete the cycles whether the traffic is endocytic, ER-Golgi traffic or granule movements. Coordinated regulation of both motors implies that there may be a central control point. A membrane protein such as kinectin or an accessory factor(s) involved in both kinesin- and cytoplasmic dynein-driven organelle movements constitute the natural focus for regulation of both motors and putative linkages to other cellular processes.

4. Kumar, J., Yu, H. and Sheetz, M.P. (1995) Kinectin, an essential Anchor for kinesin-driven vesicle motility. Science, 24 March 1995; 267(5205):1834-1837. DOI: 10.1126/science.7892610

The membrane anchor for the molecular motor kinesin is a critical site involved in intracellular membrane trafficking. Monoclonal antibodies specific for the cytoplasmic surface of the chick brain microsomes were used to define proteins involved in microtubule-dependent transport. One of four antibodies tested inhibited plus-end-directed vesicle motility by approximately 90 percent even as a monovalent Fab fragment and reduced kinesin binding to vesicles. This antibody bound to the cytoplasmic domain of kinectin, an integral membrane protein of the endoplasmic reticulum that binds to kinesin. Thus, kinectin acted as a membrane anchor protein for kinesin-driven vesicle motility.

3. Yu, H., Nicchitta, C.V., Kumar, J., Toyoshima, I. and Sheetz, M.P. (1995) Characterization of kinectin, a kinesin binding protein: primary sequence and N-terminal topogenic signal analysis. Molecular Biology of the Cell, 6(2):171-183.

Kinectin is a kinesin-binding protein (Toyoshima et al., 1992) that is required for kinesin based motility (Kumar et al., 1995). A kinectin cDNA clone containing a 4.7-kilobase insert was isolated from an embryonic chick brain cDNA library by immunoscreening with a panel of monoclonal antibodies. The cDNA contained an open reading frame of 1364 amino acids encoding a protein of 156 kDa. A bacterially expressed product of the full length cDNA bound purified kinesin. Transient expression in CV-1 cells gave an endoplasmic reticulum distribution that depended upon the N-terminal domain. Analysis of the predicted amino acid sequence indicated a highly hydrophobic near Nterminal stretch of 28 amino acids and a large portion (326-1248) of predicted a helical coiled coils. The 30-kDa fragment containing the N-terminal hydrophobic region was produced by cell-free in vitro translation and found to assemble with canine pancreas rough microsomes. Cleavage of the N terminus was not observed confirming its role as a potential transmembrane domain. Thus, the kinectin cDNA encodes a cytoplasmic-oriented integral membrane protein that binds kinesin and is likely to be a coiled-coil dimer.

2. Toyoshima, I., Yu, H., Steuer, E.R. and Sheetz, M.P. (1992) Kinectin, a major kinesin-binding protein on ER. Journal of Cell Biology, 118(5):1121-1131.

Previous studies have shown that microtubule-based organelle transport requires a membrane receptor but no kinesin-binding membrane proteins have been isolated. Chick embryo brain microsomes have kinesin bound to their surface, and after detergent solubilization, a matrix with an antibody to the kinesin head domain (SUK-4) (Ingold et al., 1988) bound the solubilized kinesin and retained an equal amount of a microsome protein of 160-kD. Similarly, velocity sedimentation of solubilized membranes showed that kinesin and the 160-kD polypeptide cosedimented at 13S. After alkaline treatment to remove kinesin from the microsomes, the same 160-kD polypeptide doublet bound to a kinesin affinity resin and not to other proteins tested. Biochemical characterization localized this protein to the cytoplasmic face of brain microsomes and indicated that it was an integral membrane protein since it was resistant to alkaline washing, mAbs raised to chick 160-kD protein demonstrated that it was absent in the supernatant and concentrated in the dense microsome fraction. The dense microsome fraction also had the greatest amount of microtubule-dependent motility. With immunofluorescence, the antibodies labeled the ER in chick embryo fibroblasts (similar to the pattern of bound kinesin staining in the same cells) (Hollenbeck, P. J. 1989. J. Cell Biol. 108:2335-2342), astroglia, Schwann cells and dorsal root ganglion cells but staining was much less in the Golgi regions of these cells. Because this protein is a major kinesin-binding protein of motile vesicles and would be expected to bind kinesin to the organelle membrane, we have chosen the name, kinectin, for this protein.

1. Yu, H., Toyoshima, I., Steuer, E.R. and Sheetz, M.P. (1992) Kinesin and cytoplasmic dynein binding to brain microsomes. Journal of Biological Chemistry, 5 October 1992; 267(28):20457-20464.

Movement of cellular organelles in a directional manner along polar microtubules is driven by the motor proteins, kinesin and cytoplasmic dynein. The binding of these proteins to a microsomal fraction from embryonic chicken brain is investigated here. Both motors exhibit saturation binding to the vesicles, and proteolysis of vesicle membrane proteins abolishes binding. The maximal binding for kinesin is 12 +/- 1.7 and 43 +/- 2 pmol per mg of vesicle protein with or without 1 mM ATP, respectively. The maximal binding for cytoplasmic dynein is 55 +/- 3.8 and 73 +/- 3.7 pmol per mg of vesicle protein with or without ATP, respectively. These values correspond to 1-6 sites per vesicle of 100-nm diameter. The nonhydrolyzable ATP analog, adenyl-5'yl imidodiphosphate (AMP-PNP), inhibited kinesin binding to vesicles but increased kinesin binding to microtubules. An antibody to the kinesin light chain also inhibited vesicle binding to kinesin. In the absence but not presence of ATP, competition between the two motors for binding was observed. We suggest that there are two distinguishable binding sites for kinesin and cytoplasmic dynein on these organelles in the presence of ATP and a shared site in the absence of ATP.

Book

8. Song, Z., Shanmugam, M.K., Yu, H., and Sethi, G. (2016) Chapter 17: Butein and Its Role in Chronic Diseases. Anti-inflammatory Nutraceuticals And Chronic diseases; Springer (ISBN 978-3-319-41334-1)

7. Yu, H., and Ananthanarayanan, A. (2013) Introduction to Cellular and Tissue Engineering (CTE). In Yu, H., Abdul Rahim, N.A. (editors), Imaging in Cellular and Tissue Engineering, CRC Press-Taylor & Francis Group, ISBN: 9781439848036.

6. Yu, H., and Abdul Rahim, N.A. (2013) Imaging in Cellular and Tissue Engineering, CRC Press-Taylor & Francis Group, ISBN: 9781439848036.

5. Ananthanarayanan, A., Tucker-Kellogg, L., Narmada, B.C., Venkatraman, L., Abdul Rahim, N.A., Wang, Y., Kang, A.C.H., and Yu, H. (2010) Systems Biology in Biomaterials and Tissue Engineering. In Ducheyne, P., Healy, K.E., Hutmacher, D.W., Grainger, C.J., Kirkpatrick, J. (editors) Comprehensive Biomaterials, volume 5, pages 178-187. Elsevier. ISBN: 978-0-08-055302-3.

4. Yue, Z.L., Lou, Y.R., Rahim, N.A.A. and Yu, H. (2009) Controlling Cellular Niche in Scaffold Designs for Epithelial Tissue Engineering. In Khang, G. (editor) The Handbook of Intelligent Scaffold for Regenerative Medicine, Singapore: Pan Stanford Publishing, in press. Publishing date: 15 August 2011. ISBN 13: 9789814267854. ISBN 10: 9814267856.

3. Khong, Y.M., Zhang, J., Zhou, S., Cheung, C., Doberstein, K., Samper, V., and Yu, H. (2010) Novel Intra-Tissue Perfusion System for Culturing Thick Liver Tissue. In Johnson, P.C., and Mikos, A.G. (editors), Advances in Tissue Engineering Volume 1 – Angiogenesis, (Chapter 28); New York: Mary Ann Liebert, Inc. publishers, ISBN13 978-1-934854-16-7.

2. Zhu, Y.J., Ng, S.S.S., Khong, Y.M., He, L.J., Toh, Y.C., Pan, X.T., Chia, S.M., Lin, P.C., Sun, W.X., Yu, H. (2005) Multi-dimensional imaging of cell- and tissue-engineered constructs. In Yu, H., Cheng, P.C., Kao, F.J., Lin, P.C. (editors), Multi-modality Microscopy, (Chapter 14); Singapore: Hackensack, N.J.: World Scientific Publishing, ISBN# 981-256-533-7.

1. Yu, H., Cheng, P.C., Kao, F.J., Lin, P.C. (Editors). (2005). Multi-modality microscopy. Singapore: Hackensack, N.J.: World Scientific Publishing, ISBN# 981-256-533-7.

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Patents and Technology Disclosures

45. Yu, H., and Liu, Z. (2016) Preparation Procedures for Cellulosic Sponge (Native, Galactosylated and Collagen-Modified.

44. Yu, H., and Liu, Z. (2016) Preparation Procedures for Cleavable Disulfide Cellulosic Sponge (Native, Galactosylated and Collagen-Modified).

43. Tan, M.-H., Yu, H., Choudhury, Y., Toh, Y.-C., Qu, Y., and Kanesvaran, R. (2015) Functional Liver Cells from B-Lymphocytes for Testing Individual Drug-Related Hepatotoxicity and Method for Producing the Same Cells.

42. Yu, H., Iliescu, C., and Yu, F. (2015) Modular Micro-Incubator System for Perfusion Cell Culture.

41. Tasnim, F., and Yu, H. (2015) Differentiation of Human Kupffer Cells from Human Pluripotent Stem Cells.

40. Yu, H., Iliescu, C., and Tong, W.H. (2015) Constrained Spheroids- A method for immobilizing spheroids for static and perfusion cell culture.

39. Yu, H., Phan, D., Toh, Y.C., and Tasnim, F. (2014) Cost-Effective Differentiation of Hepatocyte-like Cells from Human Pluripotent Stem Cells Using Small Molecules.

38. Yu, H., and Xu, S. (2012) Translation of Qualitative Pathology to Quantitative Features for Liver Disease Classification. Licensed

37. Yu, H., Toh, Y.C., and Xing, J. (2012) Micropatterned Pluripotent Stem Cell Differentiation for In Vitro Human Development Toxicity Testing.

36. Choudhury, D., Anene-Nzelu, C., Yu, H., Toh, Y.C., Leo, H.L., and Ng, S.H. (2012) Gratings-on-a-Dish – Processing of Large-Area, Low-Cost Diffraction Gratings (Holographic Gratins on Optical Discs CD.DVD) for Cell Culture/Cell Allignment.

35. Nugraha, B., and Yu, H. (2012) Cleavable macroporus cellulosic sponge for 3D cell culture and spheroids retrieval. Licensed

34. Van Noort, D., and Yu, H. (2008) Microfluidic continuous flow device for culturing biological material.

33. Cha, J.W., Singh, V.R., Yew, E., Rajapakse, J.C., Yu, H., Nedivi, E., and So, P. (2012) High sensitivity, high throughput, deep imaging based on non-descanned multifocal multiphoton microscopes.

32. Zheng, B. X., Yu, H. (2011) Quantitative drug efficacy ranking algorithm for anti-fibrosis drug discovery.

31. Yu, H., Tai, D., He, Y., Xu, S. (2011) A method and system for determining a stage of fibrosis in a liver. Licensed

30. Choi, H., Kim, D., Sheppard, C., Singh, V.R., So, P.T.C., Yew, Y.S.E., Yu, H. (2011) High Sensitivity Temporal Focusing Widefield Multiphoton Endoscope Capable of Deep Imaging.

29. Yu, H., Tai, D., He, Y. (2009) Instantaneous virtual biopsy for liver fibrosis staging using second harmonic generation microscopy.

28. Yu, H., Zhang, S., Leo, H.L. (2009) 96-well perfusion bioreactor for in vitro drug screening.

27. Yu, H., Han, R., Du, Y. (2009) Bioactive Surface for hepatocyte-based applications. Licensed

26. Yue, Z., Wen F., Yu, H. (2009) Forming Porous Scaffold from Cellulose Derivatives. Licensed

25. Yu, H., Ong, S.M. (2008). Forming cell structure with transient linker in cage.

24. Yu, H., Leo, H.L., Xia, L. (2008) Sandwich Culture Based Stack-Plate Hepatocytes Bioreactor.

23. Yu, H., Khong, Y.M., Wen, F. (2008) Fabrication of Composite Tissue Engineering Scaffolds via Gamma-Irradiation-Induced Collagen Crosslinking.

22. Zhang, C., van Noort, D., Yu, H. (2008). Multiple Soluble Microenvironments in a Compartmentalized Cellular Microfluidic System.

21. Foo, A., Yu, H. (2008). Bioactive Scaffold with Controllable Mass Transfer Properties Independent of Mechanical Strength.

20. Yue, Z., Yu, H. (2008) Method and system for transient nano-carriers for intracellular drug delivery.

19. Yu, H., Khong, Y.M., Wen, F. (2008) Intra-tissue perfusion culture of thick tissue constructs using porous needles.

18. Yu, H., Toh, Y.C., Ng, S.S. (2008) Cell Culture Device.

17. van Noort, D., Kim, N.Y., Ying, J., Yu, H. (2008). Microfluidic delivery of reagents and samples.

16. Yu, H., Khong, Y.M., Leo, H.L. (2007) Intra-tissue perfusion live bio-imaging chamber.

15. Sun, W.X., Yu, H. (2006) Sensitive second harmonic generation microscopy for quantifying matrix-related tissue dynamics and diseases.

14. Ng, S., Yu, H. (2004) Immobilization of cells in matrix formed by biocompatible charged polymers under laminar flow conditions.

13. Yu, H, Toh, Y.C. (2004) Encapsulation of cells in biologic compatible scaffolds by coacervation of charged polymers.

12. Chia, S.M., Yu, H. (2004) Application of latent/active transforming growth factor-b1 and its activators/regulators as supplements for sustained high level of hepatocyte functions in culture.

11. Kan, S.H., Schumacher, K., Ng, S.S.S., Sun, W.X., Yu, H., Ying, J. (2005). High throughput cell-based assay, fabricated with integrated silicon and cell culture technologies, for the functional characterization and detection of drugs.

10. Khong, Y.M., Yu, H. (2004) Long-term liver slice culture using novel microneedles perfusion system.

9. Hou, H.T., Zhang, J., Yu H. (2004) Device for small scaled encapsulation of animal cells.

8. Li, J., Quek, C.H., Gan, L.M., Yu, H., Leong, K.W. (2004) Microcapsules for encapsulation of bioactive substances..

7. Yu, H. Bested, S.M., Fang, S., Ang, C.E. (2003) Cell Culture System.

6. Yu, H., Cheng E., Fang S. (2002) A novel method of culturing and harvesting anchorage-dependent cells in hollow fiber bioreactors.

5. Yu, H., Leong, K.W., Chia, S.M., Wan, A.C.A. (2001) Multi-layer cell encapsulation for tissue engineering.

4. Yu, H., Leong, K.W., Chia, S.M. (2001) A non-disruptive 3D culture and harvest system for anchorage-dependent mammalian cells.

3. Wang, S, Wan, A.C.A., Leong, K.W., Yu, H. (2001) Polymer and nerve guide conduits formed thereof.

2. Li, J., Yu, H., Leong, K.W. (2000) Injectable drug delivery systems with cyclodextrin-polymer based hydrogels.

1. Leong, K.W., Mao, H.Q., Li, J., Yu, H. (1999) Polyrotaxanes hydrogel based gene delivery system.

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Conference Proceedings (In progress)

268. Hari Singh, N., McMillian, M., Qu, Y., Ng, C.W., Zhou, Y., Yu, H., and Ananthanarayanan, A. (2017) Evaluating Reactive Acyl Glucuronides Formation from Diclofenac using a Resazurin/ Resorufin Assay with Primary Rat Hepatocytes. Society of Toxicology, Baltimore, Maryland, USA, 12-16 March 2017

267. Ananthanarayanan, A., Qu, Y., Singh, N.H., Nugraha, B., McMillian, M., and Yu, H. (2017) Hepatocyte Spheroid Cultures in Galactosylated Cellulosic Sponge for Drug DMPK and Efficacy Testing. Society of Toxicology, Baltimore, Maryland, USA, 12-16 March 2017

266. Ong, L.J.Y., Chong, L.H., Jin, L., Yu, H., Ananthanarayanan, A., Leo. H.L., and Toh, Y.-C. (2017) “A Pump-Free Microfluidic 3D Perfusion Platform For The Efficient Differentiation Of Human Hepatocyte-Like Cells,” The 26th Conference of Asian Pacific Association for the Study of the Liver: APASL Annual Meeting 2017, Shanghai, China, 15-19 February 2017

265. Yan, J., Yu, Y., Kang, J.W., Tam, Z.Y., Xu, S., Fong, E.L.S., Song, Z., Tucker Kellogg, L., So, P.T.C., and Yu, H. (2017) “A Classification Model for Non-alcoholic Steatohepatitis (NASH) Using Confocal Raman Micro-spectroscopy,” The 26th Conference of Asian Pacific Association for the Study of the Liver: APASL Annual Meeting 2017, Shanghai, China, 15-19 February 2017

264. Yu, Y., Wang, J., Ng, C.W., Xu, S., Xing, J., Wee, A., Welsch, R.E., So, P.T.C., and Yu, H. (2017) “sqFibrosis: A Fully Quantitative Classification Method to Facilitate Fibrosis Scoring Using Collagen Stains,” The 26th Conference of Asian Pacific Association for the Study of the Liver: APASL Annual Meeting 2017, Shanghai, China, 15-19 February 2017

263. Fong, E., Toh, T.B., Huynh, T.H., Chow, E., and Yu, H. (2017) “Development of an In Vitro Biobank of Patient-Derived Xenografts for Hepatocellular Carcinoma,” The 26th Conference of Asian Pacific Association for the Study of the Liver: APASL Annual Meeting 2017, Shanghai, China, 15-19 February 2017

262. Song, Z., Fan, J., Teo, J., Yu, Y., Jie, Y., Ma, Y., Fang, Y., Mo, S., Tucker-Kellogg, L., So, P., and Yu, H. (2017) “Imaging the liver regeneration process in Lifeact-GFP mice,” The 26th Conference of Asian Pacific Association for the Study of the Liver: APASL Annual Meeting 2017, Shanghai, China, 15-19 February 2017

261. Pawijit, P., Zhou, Y., and Yu, H. (2017) “Involvement of kinectin in drug induced liver injury,” The 26th Conference of Asian Pacific Association for the Study of the Liver: APASL Annual Meeting 2017, Shanghai, China, 15-19 February 2017

260. Gupta, K., Li, Q., Song, Z., Fong, E.L.S., and Yu, H. (2017) “Blebbing and budding: Early canalicular response to altered canalicular pressure in obstructive cholestasis,” The 26th Conference of Asian Pacific Association for the Study of the Liver: APASL Annual Meeting 2017, Shanghai, China, 15-19 February 2017

259. Singh, N.H., Wong, J.Y., Yan, W.H., Mettu, V.S., Qu, Y., Ngo, Q., Koh, P., Zhou, Y., Yu, H., Ananthanarayanan, A., and McMillian, M.K. (2017) “A resazurin to resorufin assay for reactive acyl glucuronides reveals a bromfenac metabolite glucuronide,” The 26th Conference of Asian Pacific Association for the Study of the Liver: APASL Annual Meeting 2017, Shanghai, China, 15-19 February 2017

258. Ngo, Q., Qu, Y., McMillian, M.K., Ananthanarayanan, A., Singh, N.H., Wong, J.Y., Ng, C.W., and Yu, H. (2017) “Inhibition of efferocytosis by mild hepatotoxicants,” Singh, N.H., Wong, J.Y., Yan, W.H., Mettu, V.S., Qu, Y., Ngo, Q., Koh, P., Zhou, Y., Yu, H., Ananthanarayanan, A., and McMillian, M.K. (2017) “A resazurin to resorufin assay for reactive acyl glucuronides reveals a bromfenac metabolite glucuronide,” The 26th Conference of Asian Pacific Association for the Study of the Liver: APASL Annual Meeting 2017, Shanghai, China, 15-19 February 2017

257. Qi, X., Xu, S., Dong, J., Wang, L., Liu, C., Zhao, J., Liu, F., Li, G., Yu, H., Wee, A., and Hou, J. (2017) “Sampling variability of liver fibrosis for assessment of cirrhotic portal hypertension: A qFibrosis approach,” The 26th Conference of Asian Pacific Association for the Study of the Liver: APASL Annual Meeting 2017, Shanghai, China, 15-19 February 2017

256. Yu, H. (2016) “Materiomic Screening of Topographical Cues That Bias Migration and Differentiation of Liver Progenitor Cells,” 2016 Tissue Engineering and Regenerative Medicine International Society- Asia Pacific Meeting (TERMIS-AP 2016), Taipei, Taiwan, 3-6 September 2016

255. Yu, H., Fang, Y. (2016) “Alternating flow co-culture system for drug metabolism study,” SIMTech EAC Annual Conference 2016, Singapore, 25 August 2016

254. Yu, H. (2016) “Imaging liver regeneration and diseases,” Next Generation Confocal Microscope for Advanced Bio-Imaging!, Singapore, 2 August 2016

253. Yu, H. (2016) “Mechanobiology perspective of obstructive cholestasis: opens the black box of intrahepatic bile canaliculi dynamics,” 2016 Southern Digestive Disease And Endoscopy Forum, Guangzhou, China, 8-10 July 2016

252. Yu, H., Gupta, K., Li, Q., Fong, L.S.E., Tang, H., Fan, J., Mo, S., Yu, Y., and Song, Z. (2016) “Mechanobiology opens the black boxes of cell responses to biomaterials,” Talk at Xiamen University, China, 6 July 2016

251. Yu, H. (2016) “Imaging liver regeneration and diseases,” Talk at Fujian Normal University, China, 1 July 2016

250. Yu, H., Gupta, K., Li, Q., Fong, E.L.S., Tang, H., Fan, J., Mo, S., Yu, Y., and Song, Z. (2016) “Mechanobiology approach to understanding causative mechanism of cellular responses to biomaterials: an example of bile canaliculi dynamics in collagen sandwich culture of hepatocytes and in vivo,” 10th World Biomaterials Congress, Montreal, Canada, 17-22 May 2016

249. Yu, H. (2016) “Acute and Sub-Acute Hepatotoxicity Testing in vitro Models,” Symposium on Non-animal Approaches to Safety & Efficacy Testing, Singapore, 25 January 2016

248. Ng, C.W., Yu, Y., Xia, L., and Yu, H. (2015) Predicting hepatic clearance of slow metabolized compound using hepatocyte sandwich perfusion system. Drug Metabolism Review, November 2015; 47(Supplement 1 Special Issue SI): 50-51. 19th North American Meeting of the International-Society-for-the-Study-of-Xenobiotics (ISSX) / 29th Meeting of the Japanese-Society-for-the-Study-of-Xenobiotics (JSSX), San Francisco, USA, 19-23 October 2014.

247. Yu, Y., Singh, N.H., Sakban, R.B., Xia, L., and Yu, H. (2015) Investigating drug-inflammation interaction of acetaminophen in hepatocytes and kupffer cells co-culture system for in vitro drug screening application. Drug Metabolism Review, November 2015; 47(Supplement 1 Special Issue SI): 154-155. 19th North American Meeting of the International-Society-for-the-Study-of-Xenobiotics (ISSX) / 29th Meeting of the Japanese-Society-for-the-Study-of-Xenobiotics (JSSX), San Francisco, USA, 19-23 October 2014.

246. Pawijit, P., Yu, Y., Zhou, Y., and Yu, H. (2015) Kinectin regulates GAP junction proteins in hepatocyte-implications in response to hepatotoxicity. Drug Metabolism Review, November 2015; 47(Supplement 1 Special Issue SI): 178-178. 19th North American Meeting of the International-Society-for-the-Study-of-Xenobiotics (ISSX) / 29th Meeting of the Japanese-Society-for-the-Study-of-Xenobiotics (JSSX), San Francisco, USA, 19-23 October 2014.

245. Iliescu, C., Yu, F., and Yu, H. (2015) “Microfluidic platforms for drug screening,” presented at 38th International Semiconductor Conference, Sinaia, Romania, 12-14 October 2015

244. Xing, X., and Yu, H. (2015) “Geometrically confined cell differentiation and migration model for human teratogen detection,” 19th European Congress on Alternatives to Animal Testing – Linz 2015, 16th Annual Congress of EUSAAT, Austria, 20-23 September 2015

243. Li, Q., Song, Z., Fan, J., Mo, S., Viasnoff, V., So, P., and Yu, H. (2015) “Mechanobiology studies of the tissue dynamics for engineering long bile canaliculi,” 7th Models of Physiology and Disease - Physiology Symposium 2015, Singapore, 21-22 September 2015

242. Li, Q., Song, Z., Fan, J., Mo, S., Viasnoff, V., So, P., and Yu, H. (2015) “Mechanobiology studies of the tissue dynamics for engineering long bile canaliculi,” The 8th Asian-Pacific Conference on Biomechanics (AP Biomech 2015), Sapporo, Japan, 16-19 September 2015

241. Yu, H. (2015) “Challenges and innovations for compound safety testing applications with scalable perfusion-based cell-culture devices,” Microfluidics and Diagnostics – Moving Microfluidic Applications from Lab to Market: Challenges & Solutions, Singapore, 14 July 2015

240. Yu. H. (2015) “Spatial and temporal morphological markers for liver regeneration and chronic liver diseases,” 7th WACBE World Congress on Bioengineering (WACBE2015), Singapore, 6-8 July 2015

239. Yu, H. (2015) “Progress in identifying image-based markers of liver cancer derived from non-alcoholic fatty liver diseases”, SMART BioSyM Workshop – Workshop on Metastatic Cancer, Singapore, 25 June 2015

238. Yu, H. (2015) “Seeing is believing: imaging the dynamic processes in liver regeneration”, MBI Weekly Meeting, Singapore, 24 June 2015

237. Ananthanarayanan, A., Nugraha, B., Qu, Y., and Yu, H. (2015) “Cleavable cellulosic sponge for 3D culture and harvest of liver cells,” The 5th Asian Biomaterials Congress (ABMC5), Taipei, Taiwan, 6-9 May 2015.

236. Yu, H. (2015) “Tissue engineered in vitro liver models for testing of drugs, pathogens, and prospects for testing food, TCM drugs, environmental toxins and cosmetics,” NUS Research Institute in Soochow Industry Park for industry audiences, Shanghai, China, 9 April 2015

235. Yu, H. (2015) “Controlling cell-cell and cell matrix interaction for engineering in vitro toxicity testing models and bioartificial liver support system,” Chinese Academic of Science, Institute of Biochemistry and Cell Biology, Shanghai, China, 8 April 2015

234. Yu, H. (2015) “Reconstitution of cell dynamics or biomolecular networks in vitro/in silico”, the A*STAR-JST Joint Workshop on "Development of fundamental technology for biodevices enabling dynamic analysis and control of cells", Singapore, 12-13 January 2015

233. Low, L., Chan, C.Y., Chen, J., Yang, H., Lee, C., Yu, H., Wenk, M., and Yap, H.K. (2014) “IL13-induced hepatic cholesterol transport defect in rat model of minial change nephrotic syndrome (MCNS)”, The American Society of Nephrology (ASN) Kidney week 2014, Philadelphia, USA, 11-16 November 2014

232. Yu, H. (2014) “Mechanobiology study of bile excretion enables innovative strategy for engineering bile collection device for drug testing applications”, The 1st International Workshop on Multiscale Mechanobiology (IWMM 2014), Hong Kong, 15-18 May 2014

231. Yu, H. (2013) “Interface structures and functions for Organs-on-Chip”, Lab-on-a-Chip Asia, Singapore, 12-13 November 2013

230. Toh, Y.C., Xing, J., Xu, S., and Yu, H. (2013) “A micropatterned human embryonic stem cell model for in vitro human developmental toxicity testing”, MicroTAS 2013, Freiburg, Germany, 27-31 October 2013.

229. Yu, H. (2013) ““科技、创新与技术”或“批判性思维的重要”或自拟” Chinese Studies in Chinese Enrichment Lecture (中国通识深广讲堂), Hwa Chong Institution, Singapore, 15 August 2013

228. Yu, H. (2013) “Academic-Industry Partnership to Support Drug Development”, Temasek Polytechnic Annual Industry Networking Event Seminar, Partners-in-Science: Achieving Commercial Success through Better Quality, Safety & Efficacy, Singapore, 23 July 2013

227. Yu, H. (2013) “Optical detection of inflammation and disease”, Research Innovation in Infectious and Inflammatory Diseases, Singapore, 8-9 July 2013

226. Anene-Nzelu, C.G., Choudhury, D., Li, H., Toh, Y.-C., Ng, S.H., Leo, H.L., and Yu, H. (2013) “Gratings on a dish: a scalable cell alignment substrate on optical media”, ASME 2013 Summer Bioengineering Conference, Sunriver, Oregon, United States of America, 26-29 June 2013.

225. Xu, S., Wee, A., and Yu, H. (2013) “qFirbosis – a New Tool for Quantitative Characterization of Liver Fibrosis or Tissue Regeneration”, TERMIS-EU, Istanbul, Turkey, 17-20 June 2013.

224. Yu, H. (2013) “Quantitative Phenotypic Markers to Monitor Liver Regeneration Failure”, TERMIS-EU, Istanbul, Turkey, 17-20 June 2013.

223. Xu, S., and Yu, H. (2013) “Capsule Index: morphology and texture based quantification of liver fibrosis from the Glisson’s capsule”, 23rd Conference for Asian Pacific Association for the Study of the Liver (APSAL 2013), Singapore, 6-9 June 2013.

222. Xu, S., Wang, Y., Wee, A., Hou, J., and Yu, H. (2013) “Quantitative characterization of changes in collagen patterns for liver fibrosis assessment”, 23rd Conference for Asian Pacific Association for the Study of the Liver (APSAL 2013), Singapore, 6-9 June 2013.

221. Toh, Y.-C., Xing, J., and Yu, H. (2013) “Spatially-patterned human embryonic stem cell differentiation and migration for developmental toxicity testing”, AsiaCord 2013, Kobe, Japan, 19-20 April 2013.

220. Yu, H. (2013) “Organ-on-Chip: a biologist's perspective,” SIMTech Microfluidics Seminar 2013, 6 March 2013, Singapore

219. Yu, H. (2012) “Translating fundamental liver biology and pathology into applications”, International Conference Cellular & Molecular Bioengineering (ICCMB3), 10-12 December 2012, Singapore

218. Chooi, K.F., Phang, G.S.S., Toh, A.H.H., Rashidah, S., Tai, D., Yu, H. (2012) “Assessment of Liver Fibrosis in the Rat”,63rd AALAS National Meeting, American Association of Lab Animal Science meeting, 4-8 November 2012, Minneapolis, USA

217. Yu, H. (2012) Tissue Informatics on Liver Fibrosis. The 1st Singapore-Korea Joint Workshop for Innovative Biomedicine, Singapore, 22 June 2012.

216. Yu, H., and Sheetz, M. (2012) Nanomedicine roadmap, West China Medical School of Sichuan University, Sichuan, China, 8 June 2012.

215. Wang, Y., Toh, Y.C., Li, Q., Zheng, B., Nugraha, B., and Yu, H. (2012) Accelerated repolarization of hepatocytes population with mechanical compaction. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

214. Ananthanarayanan, A., and Yu, H. (2012) Spheroid model of liver cells for Hepatitis C infections. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

213. Hemant, V.U., and Yu, H. (2012) Braille for cells: deciphering topographic cues for cell behavior. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

212. Xing, J., Toh, Y.C., Poh, J., and Yu, H. (2012) Development of stem cell-based models for in vitro toxicity of xenobiotics. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

211. Li, Q., Robens, J. M. and Yu, H. (2012) Micro pillar array maintains primary hepatocyte’s polarity through constraining cell spreading. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

210. Nugraha, B., and Yu, H. (2012) Cellulosic hydrogel Sponge for cell-dense 3D culture platform. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

209. Ng, I.C., and Yu, H. (2012) Kinectin facilitates chemotactic migration of mesenchymal-like cells by stabilizing leading protrusions. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

208. Peng, Q., Kang, C.H., Cha, J.W., So, P.T.C., and Yu, H. (2012) Integration of Multi-Focal Multi-Photon Microscope and Second Harmonic Generation for 3D High-Resolution Imaging of Liver Fibrosis. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

207. Tee, Y.H., Heng, J.K., Zhang, X., Yeap, S.H., Ng, I.C., Shazib, M.A., and Yu, H. (2012) Physical and chemical cues elicit a unified endoplasmic reticulum response to govern cell attachment. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

206. Tong, W.H., Zhang, S., Iliescu, C., and Yu, H. (2012) Mechanically Improved RoboTox – A Robust and high throughput hepatotoxicity drug testing platform with hepatocytes spheroids. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

205. Venkatraman, L., Yu, H., Tucker-Kellogg, L. (2012) Thrombospondin-1 induces sinusoidal endothelial cell defenestration. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

204. Xia, L., Sakban, R. B., Hong, X., Qu, Y., and Yu, H. (2012) 3D in vitro hepatocyte model on RGD-galactose hybrid membrane for drug hepatotoxicity screening. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

203. Xu, S., So, P., Rajapakse, J., and Yu, H. (2012) Surface Quantification of Liver Fibrosis: From Microscopy to Endoscopy. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

202. Zhang, W., Sakban, R., Nugraha, B., Hong, X., Jia, R., Xia, L., and Yu, H. (2011) Modulation of cryochrome P450 enzyme function and expression by piperine in cellulosic sponge system. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

201. Zhu, L., Tong, W. H., Toh, Y. C., Choudhury, D., Wang, Z., Iliescu, C., and Yu, H. (2012) Enhanced micro-feature on glass silicon microfluidic channel of 3D hepatocyte culture device. The 9th World Biomaterials Congress, Chengdu, China, 1-5 June 2012.

200. Yu, H. (2012) Mechanobiology in Liver Tissue Engineering. International Workshop on Tissue Engineering 2012, Tsinghua University, Beijing, China 30th May 2012.

199. Yu, H. (2012) Novel Technique of Liver Biopsy – Surface Quantification. 5th FuRui Liver Fibrosis Forum, Guangzhou, China 25-26 May 2012.

198. Wang, J., Tucker-Kellogg, L., Ng, I.C., Jia, R., Thiagarajan, P.S., White, J., and Yu, H. (2012) The Self-Limiting Dynamics of TGF-β Signaling In Silico and In Vitro: A Novel Mechanism of PPM1A Feedback. 16th Annual International Conference on Research in Computational Molecular Biology RECOMB 2012, Barcelona, Spain, 21-24 April 2012.

197. Venkatraman, L., Chia, S.M., Narmada, B.C., Poh, L.S., White, J.K., Bhowmick, S.S., Dewey, C.F., So, P.T., Yu, H., and Tucker-Kellogg, L. (2011) PLasmin antagonizes positive feedback between TGF-β1 and TSP1: Steady states and dynamics. Biophysical Society The 56th Annual Meeting, San Diego, California, USA, 25-29 February 2012.

196. Chua, H.E., Bhowmick, S.S., Tucker-Kellogg, L., Wang, Y., Dewey, C.F. Jr and Yu, H. (2012) PANI: An Interactive Data-driven Tool for Target Prioritization in Signaling Networks. IHI 2012 : 2nd ACM SIGHIT International Health Informatics Symposium, Miami, USA, 28-30 January 2012. Proceedings of the 2nd ACM SIGHIT International Informatics Symposium, 2012:851-854.

195. Chua, H.E., Bhowmick, S.S., Tucker-Kellogg, L., Zhao, Q., Dewey, C.F. Jr and Yu, H. (2012) In Silico Identification of Endo 16 Regulators in the Sea Urchin Endomesoderm Gene Regulatory Network. IHI 2012 : 2nd ACM SIGHIT International Health Informatics Symposium, Miami, USA, 28-30 January 2012. Proceedings of the 2nd ACM SIGHIT International Informatics Symposium, 2012:131-140.25.

194. Seah, B.-S., Bhowmick, S.S., Dewey Jr. C.F., and Yu, H. (2012) “FUSE: A system for data-driven multi-level functional summarization of protein interaction networks.” IHI 2012: 2nd ACM SIGHIT International Health Informatics Symposium, Miami, USA, 28-30 January 2012. Proceedings of the 2nd ACM SIGHIT International Informatics Symposium, 2012:847-850.

193. Lin, J., Lu, F., Zheng, W., Yu, H., Sheppard, C., and Huang, Z. (2012) “An integrated coherent anti-Stokes Raman scattering and multiphoton imaging technique for liver disease diagnosis.” Multiphoton Microscopy in the Biomedical Sciences, San Francisco, USA, 22-24 January 2012. Published in Progress in Biomedical Optics and Imaging – Proceedings of SPIE, 8226:-, article number 822625. DOI: 10.1117/12.908200

192. Yu, H. (2012) Trends in liver engineering for complex tissue regeneration applications. The 4th International Conference on The Development of Biomedical Engineering – Regenerative Medicine Conference, Ho Chi Minh City, Vietnam, 8-12 January 2012.

191. Shazib, M.A., Tee, Y.H., and Yu, H. (2011) Induction of endoplasmic reticulum response by bio-physical cues govern cell attachment. The 4th International Conference on The Development of Biomedical Engineering, Ho Chi Minh City, Vietnam, 8-12 January 2012.

190. Venkatraman, L., Chia, S.M., Narmada, B., Poh, L., White, J., Bhowmick, S., Dewey, C., So, P., Yu, H., and Tucker-Kellogg, L. (2011) “Modeling the Interplay of Plasmin and Thrombospondin-1 in TGF-beta 1 Activation: A Bistable Switch in Silico and in Vitro.” Annual Meeting of the American Society for Cell Biology (ASCB), 3-7 December 2011.

189. Yu, H. (2011) “Advances in Biomedical Engineering – Liver.” Testicular Toxicology in vitro models, Baltimore, USA, 26-27 October 2011.

188. Venkatraman, L., Bhowmick, S.S., Dewey, C.F., Yu, H., and Tucker-Kellogg, L. (2011) Plasmin antagonizes positive feedback loop between TGF-β1 and TSP1: implications in liver fibrosis. EMBO – Structure and Dynamics of Protein Networks, Heidelberg, Germany, 13-16 October 2011.

187. Toh, Y. C. (2011) Differential Environmental Spatial Patterning (DESP) recreates proximal-distal axial patterns in embryonic stem cell colonies. MicoTAS 2011: The 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences, Seattle, USA, 2-6 October 2011.

186. Xu, S., Tai, D., Wee, A., Welsh, R., So, P., Yu, H., and Rajapakse, J. (2011) Automated Scoring of Liver Fibrosis through Combined Features from Different Collagen Groups. 33rd Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC ’11), Boston Marriott Copley Place Hotel, Boston, Massachusetts, USA, 30 August – 3 September 2011. Published in Conference proceedings: … Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEE Engineering in Medicine and Biology Society Conference, 2011:4503-4506, article number 6091116.

185. Chua, H.E., Bhowmick, S.S., Tucker-Kellogg, L., Dewey, C.F.Jr., and Yu, H. (2011) “PANI: A Novel Algorithm for Fast Discovery of Putative Target Nodes in Signaling Networks.” and “FUSE: Towards Multi-Level Functional Summarization of Protein Interaction Networks.” ACM Conference on Bioinformatics, Computational Biology and Biomedicine 2011 (ACM-BCB), Chicago, USA, 1-3 August 2011. Published in 2011 ACM Conference on Bioinformatics, Computational Biology and Biomedicine, BCB 2011, 2011:284-288.

184. Chua, H.E., Bhowmick, S.S., Tucker-Kellogg, L., Dewey, C.F.Jr., and Yu, H. (2011) “FUSE: Towards Multi-Level Functional Summarization of Protein Interaction Networks.” ACM Conference on Bioinformatics, Computational Biology and Biomedicine 2011 (ACM-BCB), Chicago, USA, 1-3 August 2011. (awarded Best Paper Award) Published in 2011 ACM Conference on Bioinformatics, Computational Biology and Biomedicine, BCB 2011, 2011:2-11.

183. Seah, B.-S., Bhowmick, S.S., Dewey, C.F.Jr., and Yu, H. (2011) “FUSE: a profit maximization approach for functional summarization of biological networks.” ACM Conference on Bioinformatics, Computational Biology and Biomedicine 2011 (ACM-BCB), Chicago, USA, 1-3 August 2011. Published in BMC Informatics, 13(supplement 3), article number S10. DOI: 10.1186/1471-2105-13-S3-S10

182. Nugraha, B., Yu, H. (2011) “Cellulosic Sponge Accelerates Hepatocyte Repolarization.”TERMIS EU Meeting 2011, Granada, Spain, 7-10 June 2011.

181. Nugraha, B., Yu, H. (2011) “Galactosylated Cellulosic Sponge Accelerates Hepatocyte Repolarization.” The 29th Annual Conference of the Canadian Biomaterials Society (CBS2011), Vancouver, Canada, 1-4 June 2011. Bramasta Nugraha was awarded CBS2011 Travel Award.

180. Yu, H. (2011) “TGFb signaling in liver fibrosis and regression.” 2nd Mini-Symposium on "Cell Fate Signaling" in Health and Disease, CeLS, NUS, 3 March 2011.

179. Yu, H. (2011) “Systems approach to study liver injury.” BioComplexity Symposium/Workshop, Singapore, 14-15 February 2011.

178. Lin, J., Lu, F., Zheng, W., Tai, D.C.S., Yu, H., Sheppard, C., and Huang, Z. (2011) “Multimodal nonlinear optical imaging of obesity-induced liver steatosis and fibrosis.” Multiphoton Microscopy in the Biomedical Sciences, San Francisco, USA, 23-25 January 2011. Published in Progress in Biomedical Optics and Imaging – Proceedings of SPIE, 7903:-, article number 79031V. DOI: 10.1117/12.873507

177. Singh, V.R., Rajapakse, J.C., Yu, H., and So, P.T.C. (2011) “Intensity normalization of two-photon microscopy images for liver fibrosis analysis.” Multiphoton Microscopy in the Biomedical Sciences, San Francisco, USA, 23-25 January 2011. Published in Progress in Biomedical Optics and Imaging – Proceedings of SPIE, 7903:-, article number 79030P. DOI: 10.1117/12.876246

176. Choudhury, D., van Noort, D., Iliescu, C., and Yu, H. (2011) “Fish on Chip: A Microfluidic Platform for In Vivo Drug Studies in Developing Fish Embryo.” The Second Conference on Advances in Microfluidics and Nanofluidics & Asia-Pacific International Symposium on Lab on Chip, Singapore, 5-7 January 2011.

175. Yu, H. (2011) “Controlling Extracellular Environmental Cues for Cell Shape and Functions for Applications.” The Second Conference on Advances in Microfluidics and Nanofluidics (AMN 2011) and Asian-Pacific International Symposium on Lab on Chip (APLOC 2011), Singapore, 5-7 January 2011.

174. Narmada, B.C., Venkatraman, L., Tucker-Kellogg, L., and Yu, H. (2011) “Multi-step regulation of transforming growth factor beta 1 in liver fibrosis by hepatocyte growth factor.” Keystone Symposia on Molecular and Cell Biology - TGF-beta in Immune Response: From Bench to Beside (A2),Utah, USA, 5-12 January 2011.

173. Deepak Choudhury, Danny van Noort ,Ciprian Iliescu , Hanry Yu (2010) “Fish on Chip: A Microfluidic Platform for In Vivo Drug Studies in Developing Fish Embryo.” 4th East Asian Pacific Student Workshop on Nano-Biomedical Engineering, Singapore, 15-16 December 2010.

172. Mo, X., Tan, C.H., and Yu, H. (2010) “Rapid Construction of Mechanically-confined Multi-cellular Structures using Dendrimeric Intercellular Linker.” TERMIS North America Meeting 2010, Orlando, USA, 5-8 December 2010

171. Nugraha, B., and Yu, H. (2010) “Macroporous Cellulosic Hydrogel Scaffold as 3D Hepatocyte Culture Platform.” TERMIS North America Meeting 2010, Orlando, USA, 5-8 December 2010

170. Zheng, B.X., and Yu, H. (2010) “Liver fibrosis drug discovery using high content analysis”, 4th Asian Young Researchers Conference on Computational and Omics Biology (AYRCOB 2010), Singapore, 1-3 December 2010

169. Nugraha, B., and Yu, H. (2010) “Macroporous Cellulosic Hydrogel Scaffold as 3D Hepatocyte Culture Platform.” 32nd Meeting of Japanese Society for Biomaterials, Hiroshima, Japan, 29-30 November 2010

168. Shazib, M.A., and Yu, H. (2010) “Human Embryonic Stem (hES) Cells and Induced Pluripotent Stem (iPS) Cells: a Tissue Engineer’s Perspective .” 9th Asian Congress on Oral and Maxillofacial Surgery, Kuala Lumpur, Malaysia, 25-28 November 2010

167. Yu, H. (2010) “Hepatotoxicity testing platforms for in vitro screening of xenobiotics.” Chinano Forum, Suzhou, China, 13-15 November 2010

166. Yu, H. (2010) “Biomaterials & Imaging Technologies in Liver Tissue Engineering.” Seminar at John Hopkins University, Baltimore, United States, 27 October 2010

165. Yu, H. (2010) “Microscale engineering of in vitro hepatocyte-based models.” Seminar and Round Table at Roche, Nutley, United States, 26 October 2010

164. Yu, H. (2010) “Adapting micro-engineered 3D hepatocyte models for drug testing applications.” Seminar at Johnson & Johnson, Raritan, United States, 25 October 2010

163. Yu, H. (2010) “Liver models for hepatotoxicity testing of drugs and fibrosis studies.” Physiology Symposium – “Models in Physiology and Disease”, National University of Singapore, Singapore, 2-3 August 2010

162. Tee, Y.H., Li, Q., and Yu, H. (2010) “Impact of Substrate-mediated Cell Shape Control on Liver Cell Functions and Applications.” RCE Symposium on Mechanobiology at World Congress on Biomechanics 2010, Singapore Suntec Convention Centre, Singapore, 1-6 August 2010.

161. Nugraha, B., and Yu, H. (2010) “Macroporous Cellulosic Hydrogel Scaffold for Liver Tissue Engineering.” 6th World Congress on Biomechanics, Singapore Suntec Convention Centre, Singapore, 1-6August 2010.

160. Nugraha, B., and Yu, H. (2010) “Cellulosic Hydrogel Scaffold for Liver Tissue Engineering Application.” The 5th SBE International Conference on Bioengineering and Nanotechnology, Biopolis, Singapore, 1-4 August 2010

159. Tee, Y.H., Zhang, X., Heng, J.K., and Yu, H. (2010) “Kinectin-mediated endoplasmic reticulum dynamics supports focal adhesion growth in the cellular lamella.” Gordon Research Conferences, Davidson, United States, 11-16 July 2010. Awarded Best Oral Presentation Award at 1st Graduate Scientific Congress, 25 January 2011.

158. Ng, I.C., Teo, L.Y., Lee, S.Y., and Yu, H. (2010) “Upregulation of kinectin during epithelial-mesenchymal transition promotes cell migration and invasion.” Gordon Research Conferences ‘Signaling By Adhesion Receptors’, Waterville, United States, 10-16 July 2010

157. He, Y., Yu, H., So, P.T.C. (2010) “Non-linear optical microscopy in liver fibrosis surface assessment.” 14th International Conference “Laser Optics 2010”, St. Petersburg, Russia, 28 June – 2 July 2010

156. He, Y., Yu, H., So, P. (2010) “Quantitative assessment of liver fibrosis using non-linear optical microscope across liver surface.” 43rd Annual Meeting of the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN 2010), Istanbul, Turkey, 9-12 June 2010. Published in Journal of Pediatric Gastroenterology and Nutrition, 50(supplement 2):E151-E151.

155. Choudhury, D., Zhang, C., van Noort, D., and Yu, H. (2010) “Development of a predictive physiologically based pharmacokinectic (PBPK) microfluidics chip.” International Symposium on Microchemistry and Microsystems (ISMM) 2010, Hong Kong, 28-30 May 2010

154. Choudhury, D., van Noort, D., and Yu, H. (2010) “Fish on a chip: A microfluidic platform for in vitro drug studies in developing fish embryo.” International Symposium on Microchemistry and Microsystems (ISMM) 2010, Hong Kong, 28-30 May 2010

153. Chua, H.E., Bhowmick, S.S., Forbes, C.D., Yu, H., Tucker-Kellogg, L. (2010) “In Silico Approach for Identifying Sensitive Nodes in Biological Network.” RECOMB 2010 Fourteenth International Conference on Research in Computational Molecular Biology, Lisbon, Portugal, 25-28 April 2010

152. Yu, H., So, P., Tai, D., He, Y., Xu, S., Peng, Q., Yew, E., and Sheppard, C. (2010) “Liver fibrosis research with non-linear optics.” Focus on Microscopy Conference, Shanghai, China, 28-31 March 2010

151. Yu, H. (2010) “Liver Tissue Engineering: Basic Research and their Translation into the Therapies of the Future.” The 20th Conference of the APASL, Beijing, China, 27 March 2010

150. Yu, H. (2010) “Microfabricated perfusion cell-based drug testing platforms.” 1st International Conference on MedTech Manufacturing Technologies (MedTech 2010), Singapore, 18-19 March 2010

149. Ng, I.C., Teo, L.Y., Lee, S.Y., and Yu, H. (2010) “Upregulation of 160-kDa kinectin during epithelial-mesenchymal transition promotes cell migration and invasion.” 2nd NGS Student Student Symposium, Singapore, 5 February 2010. Awarded Best Poster.

148. Nugraha, B., Yue, Z., and Yu, H. (2010) “Novel Cellulosic Hydrogel Scaffold for Liver Tissue Engineering”, 2nd NGS Student Symposium, Singapore, 5 February 2010

147. Lu, F., Zheng, W., Tai, D.C.S., Lin, J., Yu, H., and Huang, Z. (2010) “Assessment of fibrotic liver disease with multimodal nonlinear optical microscopy.” Multiphoton Microscopy in the Biomedical Sciences, San Francisco, USA, 24-26 January 2010. Published in Progress in Biomedical Optics and Imaging – Proceedings of SPIE, 7569:-, article number 75691W. DOI: 10.1117/12.843127

146. Venkatraman, L., Yu, H., Bhowmick, S.S., Forbes, D.Jr., Tucker-Kellogg, L. (2010) “The steady states and dynamics of urokinase mediated plasmin activation.” Pacific Symposium on Biocomputing 2010, The Big Island of Hawaii, 4-8 January 2010

145. Mo, X., Nugraha, B., Zhang, C., Toh, Y.C., Tan, C.H., Wang, Y., Yu, H. (2009) “Micro-Fabrication Factory of Complex Tissues.” 3rd East-Asian Pacific Student Workshop on Nano-Biomedical Engineering, Singapore, 21-22 December 2009

144. Nugraha, B., Yue, Z., Yu, H. (2009) “Novel Cellulosic Hydrogel Scaffold for Liver Tissue Engineering”, 3rd East Asian Pacific Student Workshop on Nano-biomedical Engineering, Singapore, 21-22 December 2009. 2nd winner for Oral Presentation Award

143. Nugraha, B., Yue, Z., Yu, H. (2009) “3D Cellulosic Gel for Hepatotoxicity Screening.” 31st Annual Meeting of Japanese Society for Biomaterials, Kyoto, Japan, 16-17 November 2009

142. Van Noort, D., Yu, H. (2009) “In vivo drug testing in microfluidics on Medaka fish embryo.” uTAS 2009, ICC Jeju, Jeju, Korea, 1-5 November 2009

141. Tai, D.C.S., Tan, N., Kang, C.H., Cheng, C.L., Chia, S.M., Xiao, G.F., Sun, W.X., Wee, A., Yu, H. (2009) "Fibro-C-index – A standardized quantification of liver fibrosis using second harmonic generation and two-photon microscopy.” The Liver Meeting, Boston, 30 October – 3 November 2009. Published in Hepatology, 50(4 supplement S):15A-816A.

140. Dan, Y.Y, Amer, L. Zhang, S.F., Yu, H., Wong, P.C., Lim, S.G. (2009) “Fetal Liver Progenitor Niche.” The 60th Annual Meeting of the American Association for the Study of Liver Diseases 2009, Hynes Convention Center, Boston, USA, 29 October – 3 November 2009. Published in Hepatology, 50(4 supplement S):900A-900A.

139. Chua, H.E., Bhowmick, S.S., Yu, H., Forbes Jr., C.F. (2009) “In silico approach to identify sensitive molecules as potential drug targets.” BMES 2009 Annual Fall Scientific Meeting, Pittsburgh, United States, 7-10 October 2009

138. Abdul Rahim, N.A., Iliescu, C., Kamm, R., Yu, H. (2009) “Microfluidic device captures correlation between human mesenchymal stem cell differentiation capacity and migration activity.” 2009 World Stem Cell Summit, Baltimore, Maryland, USA, 21-23 September 2009.

137. Yu, H. (2009) “Engineering in vitro drug testing platforms.” ICMAT Symposium A - Advanced Biomaterials and Regenerative Medicine (In Conjunction with 2nd Asian Biomaterials Congress), Singapore, 28 June – 3 July 2009

136. Yue, Z., Mo, X., Nugraha, B., Tan, C.H., Yu, H. (2009) “Biomaterials to Facilitate Controls of Cell-Matrix and Cell-Cell Interactions in Soft Tissue Construction.” 2nd Asian Biomaterials Congress (ABMC), Singapore, 26 - 27 June 2009

135. Xia, L., Leo, H.L., Yu, H. (2009) “Development of a novel hepatocyte sandwich culture based bioreactor for bioartificial liver assist device.” Advances in Synthetic Biology, London, England, 28-29 April 2009

134. Abdul Rahim, N. A., D Kamm, R., Yu, H. (2009) “Effects of rigidity sensing on fibrotic cellular activity.” Biomaterials Asia 2009, Hong Kong, 5-8 April 2009

133. Zhang, C., Chia, S.M., Ong, S.M., Zhang, S., van Noort, D., Yu, H. (2009) “Incorporation of TGF-β1 in a microfluidic device to enhance primary heptocyte functions,” The 5th International Conference on Microtechnologies in Medicine and Biology, Quebec City, Canada, 1-3 April 2009

132. Tai, D.C.S., Tan, N., Kang, C.H., Cheng, C.L., Chia, S.M., Xiao, G.F., Sun, W.X., Wee, A., Yu, H. (2009) “Fibro-Index: Automated, 1-step system to assess liver fibrosis stage.” Biomedical Asia 2009, Singapore, 16-19 March 2009

131. van Noort, D., Toh, Y.C., Yu, H. (2009) “A Microfludics Device for Cellular Drug Screening”. IEEE International Conference on Micro Electro Mechanical Systems (MEMS) 2009, Hilton Sorrento Palace, Sorrento, Italy, 25-29 January 2009

130. Yu, H (2008) “Precision engineering of complex internal organs: design parameters for biomaterials and device developments”. A*STAR CCO Workshop on Biomaterials: Materials in Biology and Medicine, Breakthrough and Discovery Theatrettes, Biopolis, Singapore, 15 December 2008

129. Yu, H. (2008) “Functional Systems Biology and Engineering: a process-centered approach to the solutions for liver diseases”. GPBE-Tohoku Graduate Student Conference in Bioengineering, Centre for Life Sciences (CeLS), NUS, Singapore, 9-10 December 2008

128. Nugraha, B., Yue, Z., Yu, H. (2008) “Cellulosic Scaffold for 3D Hepatocyte Culture”. NUS-Tohoku Graduate Student Conference in Bioengineering, Centre for Life Sciences (CeLS), Singapore, 9-10 December 2008

127. Tai, D.C.S., Tan, N., Chia, S.M., Xu, S.Y., Kang, A.C.H., Yu, H. (2008) “Automated Algorithm for Standard Quantification on Liver Fibrosis using Second Harmonic Generation Microscopy”. IEEE PhotonicsGlobal@Singapore 2008, SMU Conference Centre, Singapore, 8-11 December 2008. Published in 2008 IEEE PhotonicsGlobal at Singapore, IPGC 2008, 2008:312-315, article number 4781383.

126. Zhang, S., Xia, L., Leo, H.L., Yu, H. (2008) “Primary sandwich perfusion culture based on microfabricated silicon nitride membranes.” The TERMIS North America 2008 Annual Conference and Exposition, Hyatt Regency La Jolla, San Diego, California, USA, 7-10 December 2008

125. Zhou, J., Bi, C., Liu, S.C., Chng, W.J., Tay, K.G., Yu, H., Glaser, K.B., Albert, D.H., Davidsen, S.K., Chen, C.S. (2008) “Identification of core gene signature associated with synergism between ABT-869, a FLT3 inhibitor and SAHA, a HDAC inhibitor in AML with FLT3-ITD mutation.” 50th Annual Meeting of American Society of Hematology (ASH), Moscone Centre, San Francisco, CA, 6-9 December 2008. Published in Blood, 112(11):572-572.

124. Leo, H.L., Xia, L., Zhang, S., Cheng, T., Xiao, G., Tuo, X., Yu, H. (2008) “Computational fluid dynamics investigation of the effect of the fluid-induced shear stress on hepatocyte sandwich perfusion culture.” The 13th International Conference on Biomedical Engineering (ICBME 2008), Suntec Singapore International Convention and Exhibition Centre, Singapore, 3-6 December 2008. Published in 13th International Conference on Biomedical Engineering, volumes 1-3, 23(1-3):1405-1408.

123. Zheng, B.X., Chia, S.M., Tai, D.C.S., Yu, H. (2008) Image-based multi-dimensional and multi-variate profiling system of anti-fibrotic compounds. High Content Cellular Screening Workshop. Genome Institute of Singapore. Human Genome Organization, Singapore, 12-14 November 2008

122. Yu, H. (2008) “Cross-scale imaging opportunities and challenges in Singapore.” 2nd Mechanobiology Workshop 2008, Centre of Life Sciences, Auditorium 1, National University of Singapore, Singapore, 3-6 November 2008

121. Xu, S.Y, He, Y.T., Tai, D.C.S., Chang, S., Rajapakse, J.C., Yu, H. (2008) “Quantification of liver fibrosis from liver surface with second harmonic generation”. 2nd Mechanobiology Workshop 2008, Centre of Life Sciences, Singapore, 3-5 November 2008

120. Raja, A.M., Sun, W., Tai, D.C.S., Chen, C.S., Yu, H. (2008) “Collagen visualized using Second Harmonic Generation Microscopy as a Breast Cancer Staging tool.” 2nd Mechanobiology Workshop, Centre of Life Sciences, Singapore, 3-5 November 2008

119. Tai, D., Xu, S., Kang, C.H., Tan, N., Chia, S.M., Cheng, C.L., Yu, H. (2008) “Standardized Quantification for Liver Fibrosis Assessment Using Second Harmonic Generation Microscopy.” 2008 Frontiers in Optics (FiO)/Laser Science XXIV (LS) Conference, Rochester Riverside Convention Centre, Rochester, New York, USA, 19-23 October 2008

118. Yu, H. (2008) “3D Cellular Models for Hepatotoxicity” BIT’s 6th Annual Congress of International Drug Discovery Science and Technology (IDDST), Loong Palace Hotel & Resort, Beijing, China, 18-22 October 2008

117. Chua, E.H., Koo, A.J.A., Bhowmick, S.S., Yu, H., Dewey, C.F.Jr. (2008) “Quantitative modeling of ischemia/reperfusion injury in heart and liver”. , St. Louis, USA, 2-4 October 2008

116. van Noort, D., Toh, Y.C., Lim, T.C., Yu, H. (2008)“3D cell cultures in multi-channel microfluidics for drug screening.” SBE’s 4th International Conference on Bioengineering and Nanotechnology, University College Dublin, Ireland, 22-24 August 2008

115. Chia, S.M., Kuan, F.Y., Tan, N., Teo, S.T., Venkatraman, L., Bhowmick, S., Yu, H. (2008) “TGFß1 Homeostasis is Important for Liver Fibrosis Resolution.” World Congress of Pediatric Gastroenterology, Hepatology and Nutrition, Iguassu Falls, Brazil, 16-20 August 2008

114. Tan, N., Chia, S.M., Teo, S.T., Tai, D.C.S., Kang, A.C.H., Cheng, C.L., Chiang, L.W., Xiao, G.F., Yu, H. (2008) “Effects of Phytosomal Silybin (Siliphos) on Bile Duct Ligation-Induced Liver Fibrosis in Rats – Is Cirrhosis Really Reversible?” World Congress of Pediatric Gastroenterology, Hepatology and Nutrition, Iguassu Falls, Brazil, 16-20 August 2008

113. Tai, D.C.S., Tan, N., Chia, S.M., Xiao, G.F., Kang, C.H., Sun, W.X., Yu, H. (2008) “Quantification of Liver Fibrosis using Second Harmonic Generation Laser Microscopy.” World Congress of Pediatric Gastroenterology, Hepatology and Nutrition, Iguassu Falls, Brazil, 16-20 August 2008

112. Nugraha, B., Yue, Z., and Yu, H. (2008) “Mitochondrial Drug Delivery System for Cancer Treatment: A Preliminary Study.” Tohoku-NUS Student Joint Symposium, Tokyo & Sendai, Japan, 10-12 May 2008

111. Xia, L., Du, Y., Leo, H.L., Yu, H. (2008) “Maintenance of hepatocyte polarity in synthetic sandwich culture.” 5th Tampere Tissue Engineering Symposium, Tampere, Finland, 23-25 April 2008

110. Tai, D.C.S., Tan, N., Chen, C.L., Chia, S.M., Xiao, G.F., Sun, W.X., Yu, H. (2008) “Standardized quantification on liver fibrosis using second harmonic generation and two-photon microscopy.” Focus on Microscopy, Osaka-Awaji, Japan, 13-16 April 2008

109. Kang, C.H., Tai, D.C.S., Xu, S., Tan, N., Chia, S.M., and Yu, H. “Quantification of collagen in second harmonic imaging of liver fibrosis”. Focus on Microscopy, Osaka, Japan, 13-16 April 2008

108. Leo, H.L., Lim, A.L.W., Yi, D.A.M., Lei, X., Arooz, T., Yue, Z., Yu, H. (2008) “Perfusion based 3D culture for in vitro drug testing.” 3rd Materials Research Society of Singapore Conference on Advanced Materials (ICAM), Singapore, 18-21 February 2008

107. Yue, Z., Der, A.T.E., Yu, H. (2008) “Therapeutic nanocarriers for controlled intracellular drug release.” 3rd Materials Research Society of Singapore Conference on Advanced Materials (ICAM), Singapore, 18-21 February 2008

106. Chia, S.M., Kuan, F.Y., Tan, N., Venkatraman, L., Bhowmick, S., Yu, H. (2008) “TGF-ß homeostasis is important for liver fibrosis resolution.” Keystone Symposium on TGF-ß Family in Homeostasis and Disease, Santa Fe, New Mexico, USA, 3-8 February 2008

105. van Noort, D., Zhang, C., Toh, Y.C., Ong, S.M., Yu, H. (2008)“3D cell cultures in microfluidics for drug discovery.” SBE’s 1st International Conference on Stem Cell Engineering, Coronado Island, CA, USA, 20-23 January 2008

104. Kumar PR, A., Prasad, T., Xiao, G., Kumary, TV, Yu, H. (2008) “Three dimensional in vitro tissue models for toxicity studies.” International Conference on Advances in Bioresorbable Biomaterials for Tissue Engineering, Singapore, 5-6 January 2008

103. Wen, F., Khong, Y.M., Du, Y., Yue, Z., Teoh, S.H., Yu, H. (2008) “Surface modification of bulky tissue engineering scaffold through gamma irradiation” International Conference on Advances in Bioresorbable Biomaterials for Tissue Engineering, Singapore, 5-6 January 2008

102. Chia, S.M., Mao, H.Q., Yu, H. (2008) “Sustained presentation of transforming growth factor β 1 (TGF-β1) to encapsulated hepatocytes mimicking the stimulatory effects of 3D co-culture.” International Conference on Advances in Bioresorbable Biomaterials for Tissue Engineering, Singapore, 5-6 January 2008

101. Tai, D.C.S., Kang, C.H., Chia, S.M., Tan, N., Xiao, G., Yu, H. (2008) “Quantification of liver fibrosis using second harmonic generation laser microscopy.” International Conference on Advances in Bioresorbable Biomaterials for Tissue Engineering, Singapore, 5-6 January 2008

100. Zhao, D., Yu, H. (2008) “Rapid engineering of multi-cellular aggregates with novel polymeric intercellular linker.” International Conference on Advances in Bioresorbable Biomaterials for Tissue Engineering, Singapore, 5-6 January 2008

99. Toh, Y.C., Zhang, C., Hutmacher, D.W., Yu, H. (2008) “A 3D microfluidic cell culture system (3D-mFCCS) for probing osteogenic differentiation of mesenchymal stem cells.” International Conference on Advances in Bioresorbable Biomaterials for Tissue Engineering, Singapore, 5-6 January 2008

98. Xia, L., Du, Y., Leo, H.L., Yu, H. (2008) “The relationship between hepatocyte density and polarity in synthetic sandwich culture.” International conference on Advances in Bioresorbable Biomaterials for Tissue Engineering, Singapore, 5-6 January 2008

97. Zhang, X., Heng, J.K., Yu, H. (2007) “Endoplasmic Reticulum at Cell Leading Edge Is Essential to Regulate Cell Migration.” The 47th American Society of Cell Biology (ASCB) Annual Conference, Washington Convention Center, Washington, DC, 1-5 December 2007

96. Zhou, J., Bi, C., Poon, L.F., Janakakumara, J.V., Khng, J., Yu, H., Glaser, K.B., Albert, D., Davidsen, S.K., Chen, C.C. “Overactivation of STAT pathways and overexpression of survivin confer resistance to FLT3 inhibitors and could be therapeutic targets in AML.” Annual Meeting of American Society of Hematology (ASH), Atlanta, USA, 8-11 December 2007. Published in Blood, 110(11 Part 1):699A-699A.

95. Yue, Z., Usuludn, S.B.M., Yu, H. (2007) Bio-inspired multifunctional nanoassemblies for intracellular drug delivery, International Conference on Cellular & Molecular Bioengineering, Singapore, 10-12 December 2007

94. Toh, Y.C., van Noort, D., Zhang, J., Yu, H. (2007) "A microfluidic 3D in vitro model for hepatotoxicity testing." 3rd SBE International Conference on Bioengineering and Nanotechnology, Singapore, 12-15 August 2007

93. van Noort, D., Zhang, C., Toh, Y.C., Ong, S.M., Yu. H. (2007) “3D cell cultures in microfluidics for drug discovery.” CELLutions Summit, Boston, USA, 20-23 August 2007

92. Ong, S.M., He, L., Zhou, D., Tan, C.H., Yu, H. (2007) “Transient inter-cellular polymeric linkers for cell-dense 3D culture.” CELLutions Summit, Boston, USA, 20-23 August 2007

91. Zhang, S., Du, Y., Kan, S.H., Yu, H. (2007) “A micro-fabricated collagen free sandwich hepatocyte culture for drug screening application.” CELLutions Summit, Boston, USA, 20-23 August 2007

90. Ong, S.M., He, L.J., Zhao, D., Tan, C.H., Yu, H. (2007) “Transient inter-cellular polymeric linger for 3D cell culture.” International Conference on Bioengineering and Nanotechnology (ICBN), Biopolis, Singapore, 12-15 August 2007

89. Wen, F., Khong, Y., Du, Y., Yue, Z., Teoh, S., Yu, H. (2007) “Surface Modification of Bulky Tissue Engineering Scaffold through Gamma Irradiation.” International Conference on Bioengineering and Nanotechnology (ICBN), Biopolis, Singapore, 12-15 August 2007

88. Khong, Y.M., Zhang, J., Zhou, S., Cheung, C., Dobersteini, K., Samper, V. and Yu, H. (2007) “Novel intra-tissue perfusion system for culturing thick liver tissue.” International Conference on Bioengineering and Nanotechnology (ICBN), Biopolis, Singapore, 12-15 August 2007

87. Yue, Z., Usuludn, S.B.M., Yu, H. (2007) “Bio-inspired multifunctional nanoassemblies for intracellular drug delivery.” International Conference on Bioengineering and Nanotechnology (ICBN), Biopolis, Singapore, 12-15 August 2007

86. Chia, S.M., Yu, H. (2007) “Novel cell culture supplements for sustained high level of hepatocyte functions in culture.” International Conference on Bioengineering and Nanotechnology (ICBN), Biopolis, Singapore, 12-15 August 2007

85. Toh, Y.C., Zhang, C., Zhang, J., van Noort, D., Samper, V.D., Hutmacher, D.W., Yu, H. (2007) “Maintaining 3D cellular phenotypes in microfluidic channels.” International Conference on Bioengineering and Nanotechnology (ICBN), Biopolis, Singapore, 12-15 August 2007

84. van Noort, D., Zhang, C., Toh, Y.C., Ong, S.M., Yu, H. (2007) “Cells in microfluidics: 3D-constructs for drug testing.” International Conference on Bioengineering and Nanotechnology (ICBN), Biopolis, Singapore, 12-15 August 2007

83. Jing, Z., Toh, Y.C., Khong, Y.M., Du, Y., Sun, W., Yu, H. (2007) “Capillary electrophoresis with laser induced fluorescence (CE-LIF) for sensitive detection of phase I and II metabolic functions in hepatocytes.” International Conference on Bioengineering and Nanotechnology (ICBN), Biopolis, Singapore, 12-15 August 2007

82. Zhang, S., Du, Y., Kan, S.H., Yu, H. (2007) “A micro-fabricated collagen free sandwich hepatocyte culture for drug screening application.” International Conference on Bioengineering and Nanotechnology (ICBN), Biopolis, Singapore, 12-15 August 2007

81. Mythreyi Raja, A., Yu, H. (2007) “Characterization of cancer microenvironment.” Asia Pacific Rim University "Doctoral Students' Conference" Keio University, Tokyo, Japan, 30 July – 3 August 2007

80. Lee, K.H., Magalhaes, R., Gouk, S.S., Yu, H., Kuleshova, L. (2007) “Evaluative study of non-penetrating cryoprotective additives: Effects of sugars on the attachment ability of freshly isolated rat hepatocytes.” 44th meeting of Cryobiology, Lake Louise, Canada, 28 July – 1 August 2007

79. Yu, H. (2007) “Multi-dimensional live tissue constructs imaging.” The 3rd Asian and Pacific Rim Symposium on Biophotonics (APBP) in conjunction with Biophotonics Downunder II, Cairns, Australia, 10 July 2007

78. Lau, S.H., Feng, W., Yu, H. (2007) “Virtual non invasive 3D imaging of biomaterials and soft tissue with a novel high contrast CT, with resolution from mm to sub 30 nm.” International Conference on Materials for Advanced Technologies (ICMAT), Singapore, 1-6 July 2007

77. Wohland, T., Pan, X, Yu, H. (2007) “Diffusion and flow in micro- and nano-structures measured by fluorescence correlation spectroscopy.” International Conference on Materials for Advanced Technologies (ICMAT), Singapore, 1-6 July 2007

76. Yu, H. (2007) “Introduction to cell biology.” The 2nd Global Enterprise for Micro-Mechanics and Molecular Medicine (GEM4, http://www.gem4.org) Summer School, Singapore, 26 June 2007

75. Du, Y.N., Han, R.B., Yu, H. (2007) “A novel synthetic sandwich configuration for stabilizing and culturing ‘Pre-spheroid Hepatocyte Monolayer’.” Keystone symposium “Tissue Engineering and Developmental Biology”, Snowbird, USA, 12-17 April 2007

74. Wen, F., Khong, Y.M., Du, Y.N., Yue, Z.L., Teoh, S.H., Yu, H. (2007) “Surface modification of bulky tissue engineering scaffold through gamma irradiation.” Keystone symposium “Tissue Engineering and Developmental Biology”, Snowbird, USA, 12-17 April 2007

73. Yu, H. (2007) “Engineering and probing extra-cellular microenvironment in tissue engineering.” IV Tampere Tissue Engineering symposium, Institute for Regenerative Medicine University of Tampere, Finland, 12-14 March 2007

72. Chia, S.M., Kuan, F.Y., Yu, H. (2007) “Thrombospondin-1 is a Key TGF-β1 activator for hepatic stellate cells in liver fibrosis.” Keystone symposium “Fibrosis”, Tahoe City, USA, 11-15 March 2007

71. Pan, X., Toh, Y.C., Khong, Y.M., Yu, H., Shi, X., Korzh, V. and T. Wohland, "Flow profile in microchannels and microvessels.”Biophysical Journal (2007). Bethesda: Biophysical Society. 51st National Meeting of the Biophysical Society, Convention Center, Baltimore, MD, United States, 3-7 March 2007

70. Magalhaes, R., Kuleshova, L., Wang, X., Gouk, S.S., Ten, C.M., Lee, K.H., Yu, H. (2006) “Study on low temperature preservation of self-assembled cellular spheroids by vitrification.” UT Symposium on Nanobio Integration. Nano-Bio, Tokyo, Japan, 4-7 December 2006

69. Toh, Y.C., Khong, Y.M., Chang, S., Samper, V.D., Hutmacher, D.W., Yu, H. (2006) “Maintaining 3D cellular phenotypes in microfluidic channels.” UT Symposium on Nanobio Integration. Nano-Bio, Tokyo, Japan, 4-7 December 2006

68. Ng, S.S.S., Han, R.B., Leo, H.L., Yu, H. (2006) “Perfusion with excretory function promotes the long-term functional maintenance of hepatocytes.” UT Symposium on Nanobio Integration. Nano-Bio, Tokyo, Japan, 4-7 December 2006

67. Yu, H. (2006) “Engineering and imaging extra-cellular environment.” UT Symposium on Nanobio Integration. Nano-Bio, Tokyo, Japan, 4-7 December 2006

66. Yu, H. (2006) “Imaging cellular niche.” 9th International Conference on Optics Within Life Science (OWLS9), National Yang-Ming University, Taipei, Taiwan, 26-29 November 2006

65. Toh, Y.C., Khong, Y.M., Chang, S., Samper, V.D., Hutmacher, D.W., Yu, H. (2006) “Maintaining 3D cellular phenotypes in microfluidic channels.” A*STAR Graduate Academy student symposium, Singapore, 11 October 2006

64. Ng, S.S.S., Han, R.B., Leo, H.L., Yu, H. (2006) “Perfusion with excretory function promotes the long-term functional maintenance of hepatocytes.” A*STAR Graduate Academy student symposium (Best Poster Award), Singapore, 11 October 2006

63. Ng, S.S.S., Han, R.B., Yu, H. (2006) “Immediate-overlay sandwich perfusion sustains hepatocyte polarity and functions.” Asian Symposium for Biomedical Materials, Jeju Island, Korea, 20-23 August 2006

62. Ong, S.M., Yu, H. (2006) “Engineering 3-D cellular constructs using inter-cellular polymeric linkers.” 7th Asian Symposium for Biomedical Materials, Jeju Island, Korea, 20-23 August 2006

61. Du, Y.N., Han, R.B., Ng, S.S., Chia, S.M., Yu, H. (2006) “Identification and stabilization of a novel 3D hepatocyte monolayer configuration for hepatocyte-based applications.” CHI conference on “Tissue Models for Therapeutic Development”, Boston, USA, 14-17 August 2006

60. Wang, X., Wu, Y.N., Chang, S., Magalhaes, R., Yu, H., Tang, H.H., Kuleshova, L. (2006) “Porcine model: Vitrification of microencapsulated hepatocytes following an optimized isolation procedure.” CRYO2006, Hamburg, Germany, 24-27 July 2006

59. Pan, X., Yu, H., Wohland, T. (2006) "Flow profile measurements in microchannels using scanning fluorescence correlation spectroscopy.”Asia Pacific Workshop on biological Physics, NUS, Singapore, 3-5 July 2006

58. Khong, Y.M., Chang, S., Samper, V., Yu, H. (2006) “A novel intra-tissue perfusion culture of thick liver slices.” Regenerate World Congress on Tissue Engineering and Regenerative Medicine, Pittsburgh, USA, 24-27 April 2006

57. Chia, S.M., Ten, C.M., Yu, H. (2006) “Novel cell culture supplements for enhanced hepatocyte functions.” Regenerate World Congress on Tissue Engineering and Regenerative Medicine, Pittsburgh, USA, 24-27 April 2006

56. Kumar, S., Kan. S.H., Ng. S., Yu. H. (2006) “Automated cell based high throughput screening via image analysis.” Nanobio conference, San Francisco, USA, 19-21 June 2006

55. Yu, H. (2006) “Cell and tissue imaging using ultra-short lasers.” IEICE Ultra-fast Photonics Meeting, Sophia University, Tokyo, Japan, 17 March 2006

54. Wu, Y.N., Chang, S., Yu, H., Kuleshova, L. (2005) “Study on cryopreservation of cell-biomaterial constructs by vitrification.” 42nd Annual Meeting of Society for Cryobiology, Minneapolis, USA, 24-27 July 2005

53. Han, R.B., Ng, S.S.S., Yu, H. (2005) “Effect of mass transport process across the top layer of sandwich construct to hepatocytes in perfusion sandwich culture.” Tissue Engineering Society International, Shanghai, China, 22-26 October 2005

52. Ng, S.S.S, Han, R.B., Hunziker W., Yu, H. (2005) “Controlled presentation of extracellular polarity cues enhances hepatocyte repolarization and function.” Tissue Models for Therapeutics, Cambridge, USA, 29-30 September 2005

51. Toh, Y.C., Yu, H. (2005) “A 3D in vitro model for hepatocytes culture in a physiological micro-environment.” Tissue Models for Therapeutics, Cambridge, USA, 29-30 September 2005

50. Long, B., Rebsamen, B., Burdet, E., Yu, H., and Teo, C.L. (2005) “Elastic path controller for assistive devices.” 27th Annual International Conference of the Engineering in Medicine and Biology Society, IEEE-EMBS 2005, Shanghai, China, 1-4 September 2005. Published in Annual International Conference of the IEEE Engineering in Medicine and Biology – Proceedings, 7:6239-6242, article number 1615922.

49. Pan, X., Yu, H., Wohland, T. (2005) “Analysis of flow speeds and directions in a microchannel by scanning fluorescence correlation spectroscopy (sFCS).” International Biophysics Congress (by European Biophysical Societies' Association), Montpellier, France, 27 August – 1 September 2005

48. Chen, C.Z.C., Kalamegam, G., Adaikan, P.G., Yu, H. and Hutmacher, D.W. (2005) “Double-sided cell seeding on an electrospun PCL-collagen nanofiber sheet.” The 2005 European Society for Biomaterials Conference, Sorrento, Italy, 11-15 September 2005

47. Yu, H. (2005) “Challenges in liver tissue engineering?” Symposium on "New trends in biomaterials-tissue engineering", Pan Pacific Hotel, Singapore, 9 July 2005

46. Chia, S.M., Ten, C.M., Yu, H. (2005) “Novel cell culture supplements for sustained high level of hepatocyte functions in culture.” 8th International Conference on Drug-Drug Interactions: New Technologies, Clinical Approaches, Prediction and Mechanisms for the Evaluation of Drug-Drug Interactions, Seattle, USA, 15-17 June 2005

45. Chen, C.Z.C., Kalamegam, G., Adaikan, P.G., Yu, H. and Hutmacher, D.W. (2005) “Biocompatibility of electrospun PCL-collagen nanofiber scaffolds with corporal smooth-muscle cells.” The Regenerate International Conference and Exposition, Atlanta, USA, 1-4 June 2005

44. Ng, S.S.S., Toh, Y.C., Khong, Y.M., Samper, V., Yu, H. (2004) “Complex coacervating microfluidics for the immobilization of cells within micropatterned matrices.” Conference on tissue models for drug delivery, Boston, USA, 8-9 November 2004

43. Toh, Y.C., Ng, S.S.S., Khong, Y.M., Samper, V., Yu, H. (2004) “3D immobilization of primary rat hepatocytes in microfluidic channels by polyelectrolyte complex coacervation under laminar flow conditions.” Conference on tissue models for drug delivery, Boston, USA, 8-9 November 2004

42. Khong, Y.M., Samper, V., Yu, H. (2004) Novel intra tissue perfusion systems of liver slices. Conference on tissue models for drug delivery, Boston, USA, 8-9 November 2004

41. Toh, Y.C., Hutmacher, D.W., Yu, H. (2004) “Polyelectrolyte complex coacervation as a mean of improving seeding efficiency of bone marrow stromal cells in a 3D culture system.” Joint Meeting of the Tissue Engineering Society International (TESI) &the European Tissue Engineering Society (ETES), Lausanne, Switzerland, 10-13 October 2004

40. Foo, H.L., Taniguchi, A., Yu, H., Okano, T., Teoh, S.H. (2004) “A more non-invasive and efficient method of surface modifying biaxially-stretched ultra-thin PCL films fabricated without the use of toxic solvent.” 4th Asian Biomaterials Conference, Singapore

39. Chia, S.M., Lin, P.C., Koh, X.Y., Mao, H.Q., Yu, H. (2004) “Sustained presence of transforming growth factor b1 to encapsulated hepatocytes mimicking the stimulatory effects of 3D co-culture.” The first international SBE Conference on Bioengineering and Nanotechnology (ICBN), Biopolis, Singapore, 27-29 September 2004

38. Toh, Y.C., Yu, H. (2004) “Polyelectrolyte complex coacervation as a means of improving seeding efficiency of bone marrow stromal cells in a 3D culture system.” The First International SBE Conference on Bioengineering and Nanotechnology (ICBN), Biopolis, Singapore, 27-29 September 2004

37. Schumacher, K.M., Yu, H. (2004) “From the renal stem cell niche to functional tubules.” The First International SBE Conference on Bioengineering and Nanotechnology (ICBN), Biopolis, Singapore, 27-29 September 2004

36. Ho, H.T., Zhang, J., Yu, H. (2004) “Apparatus for encapsulating cells.” The First International SBE Conference on Bioengineering and Nanotechnology (ICBN), Biopolis, Singapore, 27-29 September 2004

35. Kuleshova, L.L, Ono, A., Wang, X.W., Yu, H. (2004) “Current progress in vitrification of tissue-engineered constructs.” World Congress of Cryobiology and Cryomedicine “Cryo’ 2004”, International Convention Center, Beijing, China, 15-19 July 2004

34. Zhu, J.H., Lao, X.J., Ho, H.T., and Yu, H. (2004) “Fabrication of 3-Dimensional microenvironment based on chitosan under physiological conditions for HepG2 proliferation.” 7th World Biomaterials Congress, Sydney, Australia, 17-21 May 2004. Published in Transactions – 7th World Biomaterials Congress, 2004:1507.

33. Zhang, J., Wei, H.P., Quek, C.H., Chia, S.M., and Yu, H. (2004) “Development of a capillary electrophoresis method for the quantitation of collagen methylation.” 7th World Biomaterials Congress, Sydney, Australia, 17-21 May 2004. Published in Transactions – 7th World Biomaterials Congress, 2004:1247.

32. Ng, S.S.S., Zhou, Y., Wu, Y.N., Chia, S.M., Yu, H. (2004) “Engineering extra-cellular matrices for modulation of cell behaviors.” 7th World Biomaterials Congress, Sydney, Australia, 17-21 May 2004. Published in Transactions – 7th World Biomaterials Congress, 2004:1347.

31. Quek, C.H., Li, J., Sun, T., Chan, M.L.H., Mao, H.Q., Gan, L.M., Leong, K.W., and Yu, H. (2004) “Photo-crosslinkable microcapsules formed by polyelectrolyte copolymer and modified collagen for rat hepatocyte encapsulation.” 7th World Biomaterials Congress, Sydney, Australia, 17-21 May 2004. Published in Transactions – 7th World Biomaterials Congress, 2004:340.

30. Wu, Y.N., Wang, X.W., Mao, H.Q., Yu, H. (2004) “Galactosylated collagen enhanced cell functions of hepatocytes in microcapsules.” 7th World Biomaterials Congress, Sydney, Australia, 17-21 May 2004. Published in Transactions – 7th World Biomaterials Congress, 2004:1104.

29. Chia, S.M., Lin, P.C., Koh, X.Y., Mao, H.Q., Yu, H. (2004) “Sustained present of transforming growth factor b1 (TGF-b1) to encapsulated hepatocytes mimicking the stimulatory effects of 3D co-culture.” 7th World Biomaterials Congress, Sydney, Australia, 17-21 May 2004. Published in Transactions – 7th World Biomaterials Congress, 2004:1157.

28. Zhu, J.H., Lao, X.J., Chia, S.M., Ng, S., Yu, H. (2003) “Fabrication of positively charged 3-dimensional cell-compatible supports for tissue engineering.” International Symposium on Bioprocess and Biomolecular Engineering, ECUST, Shanghai, China, 15-17 December 2003

27. Ong, L.L., Yu, H. (2003) “Assembly of translation elongation factor-1 complex on Kinectin.” 43rd Annual Meeting of The American Society for Cell Biology, San Francisco, USA, 13-17 December 2003

26. Chia, S.M., Lin, P.C., Quek, C.H., Mao, H.Q., Yu, H. (2003) “Co-Encapsulation of transforming growth factor-β1 to prolong functions of encapsulated hepatocytes.” Tissue Engineering Society International (TESI) Annual Meeting, Florida, USA, 10-13 December 2003

25. Chia, S.M., Lin, P.C., Quek, C.H., Leong, K.W., Yu, H. (2003) “TGF-β1 regulation in hepatocyte-NIH3T3 co-culture is important for the enhanced hepatocyte functions.” Tissue Engineering Society International (TESI) Annual Meeting, Orlando, USA, 10-13 December 2003

24. Lee, I.L., Wang, J., Lim, W.S., Chia, S.M., Yu, H., Leong, K.W., Mao, H.Q. (2003) “Collagen-based non-viral gene carriers.” Molecular Therapy 7 (5): S222-S222 572 Part 2

23. Kuleshova, L.L., Wang, X.W., Yu, H. (2003) “Long term preservation of microcapsulated hepatocytes.” Tissue Engineering Society International (TESI) Annual Meeting, Orlando, Florida, USA, 10-13 December 2003

22. Tan, L.S.E., Yu, H. (2003) “Influences of extracellular microenvironment on cell behaviour: A comparative approach.” The 4th Sino-Singapore Conference on Biotechnology, National University of Singapore, Singapore, 11-13 November 2003

21. Ong, L.L., Yu, H. (2003) “Assembly of translation elongation factor-1 complex on kinectin.” The 4th Sino-Singapore Conference on Biotechnology, National University of Singapore, Singapore, 11-13 November 2003

20. Kuleshova, L.L., Wang, X.W., Chua, A., Wu, Y., Yu, H. (2003) “Vitreous cryopreservation of tissue-engineered constructs.” International Conference of Cryobiomol 2003, Coimbra, Portugal, 14-18 September 2003. (Conference’s proceeding Cryobiomol 2003, p.70)

19. Ng, S., Qie, L., Chia, S.M., Cheng, P.C., Yu, H. (2002) “Study of formation of nano-fibers surrounding live cells by microscopy.” 4th International Conference on the Multi-Dimensional Microscopy and Cell and Tissue Engineering, Guangzhou, China, 27-30 November 2002

18. So, B.Y., Wong, C.H., Moochhala, S.M., Yu, H. (2002) “Antisense telomerase RNA therapy for gastric and colon cancers - an in-vitro study.” NUS-NUH combined scientific conference, Singapore, 16-17 August 2002

17. Chia, S.-M., Zhou, Y., Sun, T., Mao, H.-Q., Leong, K.W., Chen, J.-P., and Yu, H. (2002) “Issues and technologies leading to a new bio-artificial liver with microencapsulated hepatocytes.” Published in Third Smith an Nephew International Symposium – Translating Tissue Engineering into Products, 2002:71, article number MS-5.

16. Cheng, P.C., Hibbs, A.R., Yu, H., Lin, P.C., Cheng W.Y. (2002) “An estimate of the contribution of spherical aberration and self-shadowing in confocal and multi-photon fluorescent microscopy.” Microscopy Society of America, USA. Published in Microscopy and Microanalysis, 8(supplement 2):1048-1049.

15. Hsu, C.S., Girija, V., Deng, Y., Chuah, G.K., Yu, H. (2001) “Role of organic matrix in laser-induced prevention of enamel demineralization.” International Association for Dental Research (IADR) annual conference. Chiba, Japan

14. Lu, H.F., Mao, H.Q., Lim, W.S., Chia, S.M., Zhang, P.C., Yu, H., Leong, K.W. (2001) “Galactosylated PVDF membrane promotes hepatocyte attachment and functional maintenance.” Society for Biomaterials, USA

13. Chia, S.M., Lin, P.C., Quek, C.H., Xu, X., Leong, K.W., Yu, H. (2001) “3D co-culture of hepatocytes and fibroblasts for liver tissue engineering.” Hilton Head Workshop on Tissue Engineering (ET-2001), Hilton Head, USA

12. Chia, S.M., Quek, C.H., Lin, P.C., Mao, H.Q., Leong, K.W., Yu, H. (2001) “A novel 3D cell culture system for tissue engineering applications.” Hilton Head Workshop on Tissue Engineering (ET-2001), Hilton Head, USA

11. Li, J., Zhou, Z., Ni, X., Yu, H., Leong, K. (2000) “Injectable supramolecular hydrogels for controlled drug delivery.” 10th International Conference on Biomedical Engineering, Singapore, 6-9 December 2000

10. Chia, S.M., Sun, T., Xu, X., Quek, C.H., Chan, M.L.H., Li, J., Leong, K.W., Yu, H. (2000) “Optimization of hepatocyte micro-encapsulation for bio-artificial liver.” 10th International Conference On Biomedical Engineering, Singapore, 6-9 December 20001

9. Chia, S.M., X. Xu, C.A.A. Wan, K.W. Leong and H. Yu (2000) “Living cell encapsulation for tissue engineering.”Gordon Research Conference on Signal Transduction by Engineered Extracellular Matrices. Tilton School, Tilton, New Hampshire, USA, 25-30 June 2000

8. Li, J., Zhou, Z.H., Yu, H., Leong, K.W. (2000) “Injectable supramolecular hydrogel for drug and gene delivery.” 6th World Biomaterials Congress, Hawaii, USA, 15-20 May 2000

7. Wang, S., Wan, C.A.A, Mao, H.Q., Leong, K.W., Yu, H. (2000) “A biodegradable Poly(phosphoester) tube for peripheral nerve regeneration.” 6th World Biomaterials Congress, Hawaii, USA, 15-20 May 2000

6. Chia, S.M., Xu, X., Li, J., Leong, K.W., Yu, H. (2000) “Double encapsulation preserves enhanced hepatocyte functions.” 6th World Biomaterials Congress, Hawaii, USA, 15-20 May 2000

5. Er, P.N., Yu, H. (2000) “Kinectin-kinesin interaction is essential for in vivo organelle motility.” Keystone Symposium on The Dynamics Of The Cytoskeleton, Keystone, Colorado, USA, 3-9 February 2000

4. Ong, L.L., Lim, A.P.C., Yu, H. (2000) “Identification and characterization of kinectin-associated proteins.” Keystone Symposium on The Dynamics Of The Cytoskeleton, Keystone, Colorado, USA, 3-9 February 2000

3. Subramaniam, S., Huang, B., Li, J., Mao, H.Q., Leong, K.W., Yu, H., Kwang, J. (1999) “Coacervate nanospheres: a novel DNA vaccine delivery system for Mycoplasma hyppneumoniae PR1 and PR2 proteins.” The 80th annual meeting of the CRWAD (Conference of Research Workers in Animal Diseases), Chicago, USA, 7-9 November 1999

2. Chia, S.M., Li, J., Xu, X., Gao, S.J., Leong, K.W., Yu, H. (1999) “Novel hepatocyte encapsulation enhances cellular functions.” BMES/IEEE-EMBS Joint Meeting, Atlanta, Georgia, USA, 13-16 October 1999. Published in Annual International Conference of the IEEE Engineering in Medicine and Biology – Proceedings, 2:722.

1. Sheetz, M., Martenson, C., Toyoshima, I., Yu, H. (1993) “Organelle motor complex and axonal growth.” Journal of NeuroChemistry, 61: S191-S191

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Invited Talks

151. “The nuts & bolts of Peer Review,” Elsevier Publishing Campus Webinar, 7 March 2017

150. “sqFibrosis: A Fully Quantitative Classification Method to Facilitate Fibrosis Scoring Using Collagen Stains,” The 26th Conference of Asian Pacific Association for the Study of the Liver: APASL Annual Meeting 2017, Shanghai, China, 15-19 February 2017149.

149. “Materiomic Screening of Topographical Cues That Bias Migration and Differentiation of Liver Progenitor Cells,” 2016 Tissue Engineering and Regenerative Medicine International Society- Asia Pacific Meeting (TERMIS-AP 2016), Taipei, Taiwan, 3-6 September 2016

148. “Alternating flow co-culture system for drug metabolism study,” SIMTech EAC Annual Conference 2016, Singapore, 25 August 2016

147. “Imaging liver regeneration and diseases,” Next Generation Confocal Microscope for Advanced Bio-Imaging!, Singapore, 2 August 2016

146. “Mechanobiology perspective of obstructive cholestasis: opens the black box of intrahepatic bile canaliculi dynamics,” 2016 Southern Digestive Disease And Endoscopy Forum, Guangzhou, China, 8-10 July 2016

145. “Mechanobiology opens the black boxes of cell responses to biomaterials,” Talk at Xiamen University, China, 6 July 2016

144. “Imaging liver regeneration and diseases,” Talk at Fujian Normal University, China, 1 July 2016

143. “Cell Shape Models,” University of Helsinki Doctoral Thesis Opponent Invited Talk , Helsinki, Finland, 26 May 2016

142. “Mechanobiology approach to understanding causative mechanism of cellular responses to biomaterials: an example of bile canaliculi dynamics in collagen sandwich culture of hepatocytes and in vivo,” 10th World Biomaterials Congress, Montreal, Canada, 17-22 May 2016

141. “In vitro screening assay development”, Meeting with DuPont, Singapore, 20 April 2016

140. “Preamble to Chronic Tox (framing the challenge),” Workshop on Chronic Tox for Paul Carmichael’s visit, Singapore, 6 April 2016

139. “Liver Models,” Short Talks by Scientist for Paul Carmichael’s visit, Singapore, 4 April 2016

138. “Acute and Sub-Acute Hepatotoxicity Testing in vitro Models,” Symposium on Non-animal Approaches to Safety & Efficacy Testing, Singapore, 25 January 2016

137. “Engineering in vitro liver models for drug safety testing applications,” Khalifa University external examiner invited seminar, Abu Dhabi, UAE, 12 November 2015

136. “What Grant Reviewers Look Out for When Reviewing a Grant,” NHG’s Grant Preparatory Seminar, Singapore, 15 October 2015

135. “Mechanobiology studies of the tissue dynamics for engineering long bile canaliculi,” 7th Models of Physiology and Disease - Physiology Symposium 2015, Singapore, 21-22 September 2015

134. “Mechanobiology studies of the tissue dynamics for engineering long bile canaliculi,” The 8th Asian-Pacific Conference on Biomechanics (AP Biomech 2015), Sapporo, Japan, 16-19 September 2015

133. “Challenges and innovations for compound safety testing applications with scalable perfusion-based cell-culture devices,” Microfluidics and Diagnostics – Moving Microfluidic Applications from Lab to Market: Challenges & Solutions, Singapore, 14 July 2015

132. “Spatial and temporal morphological markers for liver regeneration and chronic liver diseases,” 7th WACBE World Congress on Bioengineering (WACBE2015), Singapore, 6-8 July 2015

131. “Progress in identifying image-based markers of liver cancer derived from non-alcoholic fatty liver diseases”, SMART BioSyM Workshop – Workshop on Metastatic Cancer, Singapore, 25 June 2015

130. “Seeing is believing: imaging the dynamic processes in liver regeneration”, MBI Weekly Meeting, Singapore, 24 June 2015

129. “Cleavable cellulosic sponge for 3D culture and harvest of liver cells,” The 5th Asian Biomaterials Congress (ABMC5), Taipei, Taiwan, 6-9 May 2015

128. “Tissue engineered in vitro liver models for testing of drugs, pathogens, and prospects for testing food, TCM drugs, environmental toxins and cosmetics,” NUS Research Institute in Soochow Industry Park for industry audiences, Shanghai, China, 9 April 2015

127. “Controlling cell-cell and cell matrix interaction for engineering in vitro toxicity testing models and bioartificial liver support system,” Chinese Academic of Science, Institute of Biochemistry and Cell Biology, Shanghai, China, 8 April 2015

126. “Reconstitution of cell dynamics or biomolecular networks in vitro/in silico”, the A*STAR-JST Joint Workshop on "Development of fundamental technology for biodevices enabling dynamic analysis and control of cells", Singapore, 12-13 January 2015

125. “Mechanobiology study of bile excretion enables innovative strategy for engineering bile collection device for drug testing applications”, The 1st International Workshop on Multiscale Mechanobiology (IWMM 2014), Hong Kong, 15-18 May 2014

124. “Interface structures and functions for Organs-on-Chip”, Lab-on-a-Chip Asia, Singapore, 12-13 November 2013

123. ““科技、创新与技术”或“批判性思维的重要”或自拟” Chinese Studies in Chinese Enrichment Lecture (中国通识深广讲堂), Hwa Chong Institution, Singapore, 15 August 2013

122. “Academic-Industry Partnership to Support Drug Development”, Temasek Polytechnic Annual Industry Networking Event Seminar, Partners-in-Science: Achieving Commercial Success through Better Quality, Safety & Efficacy, Singapore, 23 July 2013

121. “Optical detection of inflammation and disease”, Research Innovation in Infectious and Inflammatory Diseases, Singapore, 8-9 July 2013

120. “Quantitative Phenotypic Markers to Monitor Liver Regeneration Failure”, TERMIS-EU, Istanbul, Turkey, 17-20 June 2013

119. “Organ-on-Chip: a biologist's perspective,” SIMTech Microfluidics Seminar 2013, 6 March 2013, Singapore

118. “Translating fundamental liver biology and pathology into applications”, International Conference Cellular & Molecular Bioengineering (ICCMB3), 10-12 December 2012, Singapore

117. “Tissue Informatics on Liver Fibrosis”, The 1st Singapore-Korea Joint Workshop for Innovative Biomedicine, Singapore, 22 June 2012

116. “Nanomedicine roadmap”, West China Medical School of Sichuan University, Sichuan, China, 8 June 2012

115. “Trend Analysis for Modern Higher Education (In Chinese).” International Workshop on Tissue Engineering 2012, Tsinghua University, Beijing, China, 30 May 2012.

114. “Mechanobiology in Liver Tissue Engineering.” International Workshop on Tissue Engineering 2012, Tsinghua University, Beijing, China, 30 May 2012.

113. “Novel Technique of Liver Biopsy – Surface Quantification.” 5th FuRui Liver Fibrosis Forum, Guangzhou, China 25-26 May 2012.

112. Public Lectures on “Analysis of the 21st Century University Trends” & “Innovations, Science and Technology”. City & Culture Research Centre, Kuala Lumpur, Malaysia 11-12 May 2012.

111. “Mechanobiology PhD Program, a training to integrate biochemical signals with mechanical phenotypes to understand biological functions and translate into biomedical applications.” HKUST BME Seminar, Hong Kong, 12-13 March 2012.

110. “Trends in Liver Tissue Engineering for complex tissue regeneration applications.” Fourth International Conference on the Development of Biomedical Engineering - Regenerative Medicine Conference, Ho Chi Minh City, Vietnam, 8-10 January 2012.

109. “Advances in Biomedical Engineering – Liver.” Testicular Toxicology in vitro models, Baltimore, USA, 26-27 October 2011.

108. “Engineering Tissue Niche to Develop Innovative In Vitro Toxicity Screening Platforms.” Seminar at University of Liverpool, Liverpool, 4 July 2011

107. “Mechanobiology to study and engineer cell and tissue as a machine”, Seminar at the Hong Kong University of Science and Technology, Hong Kong, 1 June 2011

106. “Mechanobiology approach to biomaterials and tissue engineering”, Seminar at University of Hong Kong, Hong Kong, 27 May 2011

105. “Research Budgeting- A Perspective from the Investigator.” Research Preparatory Workshop, Singapore 28 April 2011

104. “TGFb signaling in liver fibrosis and regression.” 2nd Mini-Symposium on "Cell Fate Signaling" in Health and Disease, CeLS, NUS, 3 March 2011

103. “Systems approach to study liver injury.” BioComplexity Symposium/Workshop, Singapore, 14-15 February 2011.

102. “Controlling Extracellular Environmental Cues for Cell Shape and Functions for Applications.” The Second Conference on Advances in Microfluidics and Nanofluidics (AMN 2011) and Asian-Pacific International Symposium on Lab on Chip (APLOC 2011), Singapore 5-7 January 2011.

101. “Human Embryonic Stem (hES) Cells and Induced Pluripotent Stem (iPS) Cells: a Tissue Engineer’s Perspective.” 9th Asian Congress on Oral and Maxillofacial Surgery, Kuala Lumpur, Malaysia, 25-28 November 2010

100. “Hepatotoxicity testing platforms for in vitro screening of xenobiotics.” Chinano Forum, Suzhou, China, 13-15 November 2010.

99. “Biomaterials & Imaging Technologies in Liver Tissue Engineering.” Seminar at John Hopkins University, Baltimore, United States, 27 October 2010.

98. “Microscale engineering of in vitro hepatocyte-based models.” Seminar and Round Table at Roche, Nutley, United States, 26 October 2010.

97. “Adapting micro-engineered 3D hepatocyte models for drug testing applications.” Seminar at Johnson & Johnson, Raritan, United States, 25 October 2010.

96. “Liver models for hepatotoxicity testing of drugs and fibrosis studies.” Physiology Symposium – “ Models in Physiology and Disease”, National University of Singapore, Singapore, 3 August 2010.

95. “Impact of Substrate-mediated Cell Shape Control on Liver Cell Functions and Applications.” RCE Symposium on Mechanobiology at World Congress on Biomechanics 2010, Singapore Suntec Convention Centre, Singapore, 2 August 2010.

94. “Liver Tissue Engineering: Basic Research and their Translation into the Therapies of the Future.” The 20th Conference of the APASL, Beijing, China, 27 March 2010

93. “Microfabricated perfusion cell-based drug testing platforms.” 1st International Conference on MedTech Manufacturing Technologies (MedTech 2010). Session 2: Advanced and emerging technologies, cost-effective manufacturing processes, and testing and reliability of medical devices. Suntec Singapore, Singapore, 18-19 March 2010.

92. “Quantitative Control of Polarized Epithelial Cell Phenotypes via Extra-Cellular Cues.” DBS Seminar, NUS, 18 December 2009

91. “Validation of Novel Cell-based Models for In Vitro Hepatotoxicity Testing of Compounds.” Johnson and Johnson Mini-Symposium, Raritan, United States, 18 November 2009

90. “CSB of Liver Fibrosis Regression Mechanisms and Therapy.” SMA-CSB Bootcamp Mini Research Symposium, Singapore, 28 July 2009

89. “Liver fibrosis/image informatics/potential projects.” SMA-CSB Bootcamp lecture, Singapore, 29 June 2009

88. “Engineering in vitro drug platforms.” ICMAT Symposium A - Advanced Biomaterials and Regenerative Medicine (In Conjunction with 2nd Asian Biomaterials Congress), Singapore, 28 June – 3 July 2009

87. “Biomaterials to Facilitate Controls of Cell-Matrix and Cell-Cell Interactions in Soft Tissue Construction.” 2nd Asian Biomaterials Congress (ABMC), Singapore, 26 – 27 June 2009

86. “Engineering in vitro cell-based drug testing platforms”, Department of Engineering Science, Oxford University, Oxford, United Kingdom, 17 June 2009

85. “Biomaterials in tissue engineering”, Lecturing at VI Tissue Engineering Symposium, Tampere, Finland, 4 June 2009

84. “Engineering in vitro drug testing platforms”. Biomedical Engineering Forum, The University of Hong Kong (HKU), 9 April 2009

83. “A multidisciplinary approach to study function process: an example of TGFβ1 regulation in liver fibrosis regression”. Physiology Seminar, NUS, 13 March 2009

82. “Computation & Systems Biology and Mechanobiology Programs”. SMA Roadshow and recruitment trip in Tsinghua University, Peking University, Beijing Normal University, University of Science and Technology, National Centre of Nanoscience and Technology, Nankai University, Tianjin University. China, 26 February – 3 March 2009

81. “Computation & Systems Biology and Mechanobiology Programs”. SMA Roadshow and recruitment trip in Xiamen University, Wuhan University, Zhejiang University, Fudan University, and Shanghai Jiaotong University. China, 16-21 February 2009

80. “Precision engineering of complex internal organs: design parameters for biomaterials and device developments”. A*STAR CCO Workshop on Biomaterials: Materials in Biology and Medicine, Breakthrough and Discovery Theatrettes, Biopolis, Singapore, 15 December 2008

79. “Functional Systems Biology and Engineering: a process-centered on approach to the solutions for liver diseases”. GPBE-Tohoku Graduate Student Conference in Bioengineering 2008, Centre for Life Sciences (CeLS) NUS, 9-10 December 2008

78. “Cross-scale imaging opportunities and challenges in Singapore”. Session 2: Bioimaging. Second Mechanobiology Workshop 2008, Centre of Life Sciences NUS, Singapore, 3-6 November 2008

77. “Functional systems biology: examples in liver research”. Department of Gastroenterology, 1st Military Medical University, Southern Hospital, Guangzhou, China, 31 October 2008.

76. “Functional Systems Biology: a process-centered approach to the study of liver fibrosis regression”. Seminar for SMA in Tsinghua University, Beijing Normal University and Nan Kai University, Beijing, China, 20-21 October 2008

75. “3D Cellular Models for Hepatotoxicity”. Session 8: The 3D Cell Models and Supporting Technologies for Hepatotoxicity Testing of Drugs. BIT's 6th Annual Congress of International Drug Discovery Science and Technology (IDDST), Beijing, China, 18-22 October 2008

74. Guest Lecturer, “Functional Systems Biology”, CS6280 Computational Systems Biology, COM1, NUS, 7 October 2008

73. “Trends in Liver Tissue Engineering” Focus Group Meeting of NUS Tissue Engineering Program (NUSTEP), Hippocrates seminar room, DSO building, Singapore, 16 September 2008

72. “Engineered liver cell/tissue constructs, possible in vitro models for infectious agents” SMART Infectious Disease Retreat, Bintan Lagoon Resort, Lagoi, Riau, Indonesia, 3-5 August 2008

71. “3D Cellular Models for Hepatotoxicity” BIT Life Sciences’ 1st Annual World Congress of ibio2008, Hangzhou, China: New Starting Line for Decision Makers in Bio-economy Era, 18-22 May 2008

70. “Cell responses to micro- and nano- featured environments.” Engineered Surfaces for Regulating Cell Behaviour. Thursday, Biopolis, Singapore, 28 February 2008

69. “Confocal microscopy and its applications.” Science and Technology Fest 2008, Biopolis, Singapore, 31 January 2008

68. “New Development in Multi-Modal Imaging for Evaluation of Cells in 3D Bioresorbable Scaffolds”, the International Conference on Advances in Bioresorbable Biomaterials for Tissue Engineering - from Research to Clinical Applications. Marina Mandarin Hotel, Singapore, 5-6 January 2008

67. “Endoplasmic Reticulum Dynamics in Cell Migration” National Taiwan University, Taipei, Taiwan, 14 November 2007

66. “Micro-/Nano-Scale Tissue Engineering in Regenerative Medicine”, National Taiwan University, Taipei, Taiwan, 12 November 2007

65. “ER dynamics in Cell Motility and Engineering Extra-Cellular Micro-environmental Cues” The 1st Mechanobiology Workshop, National University of Singapore, Singapore, 16-18 October 2007

64. “Nanotechnology and Tissue Engineering”, keynote address, Graduate Program in Bioengineering Graduate Student Conference (GPBE) National University of Singapore, Singapore, 14 September 2007

63. “Multi-dimensional live tissue constructs imaging", The 3rd Asian and Pacific Rim Symposium on Biophotonics (APBP) in conjunction with Biophotonics Downunder II, Cairns, Australia, 10 July 2007

62. “Introduction to Cell Biology”, the 2nd Global Enterprise for Micro-Mechanics and Molecular Medicine (GEM4) Summer School, Singapore, 26 June 2007

61. “Endoplasmic Reticulum Dynamics and Liver Fibrosis Resolution”, The Centre for Reproduction, Development and Growth, University of Hong Kong, Hong Kong, 10 April 2007

60. “Challenges in Micro-/Nano-Scale Tissue Engineering in Regenerative Medicine”, Department of Mechanical Engineering, University of Hong Kong, Hong Kong, 3 April 2007

59. “Engineering and probing extra-cellular microenvironment in tissue engineering”, IV Tampere Tissue Engineering symposium, Institute for Regenerative Medicine University of Tampere, Finland, 12-14 March 2007

58. “Engineering and imaging extra-cellular environments”, Nanobio-Tokyo, University of Tokyo, Japan, 4-7 December 2006

57. “Imaging Cellular Niche”, 9th International Conference on Optics Within Life Science (OWLS9), National Yang-Ming University, Taipei, Taiwan, 26-29 November 2006

56. “Engineering Extra-Cellular (EC) Environments”, EWHA Woman University, Division of Nano Science & Department of Life Science, Seoul, Korea, 24 August 2006

55. “Immediate-Overlay Sandwich Perfusion Sustains Hepatocyte Polarity and Functions”, ASBM 7, JeJu Island, Korea, 20 August 2006

54. “Cell and Tissue Imaging Using Ultra-short Lasers”, IEICE Ultra-fast Photonics Meeting, Sophia University, Tokyo, Japan, 17 March 2006

53. "Liver cell and tissue engineering: systematic approaches to applications". NUS Tissue Engineering Program (NUSTEP) workshop on Regenerative Medicine and Tissue Engineering, Singapore, 5-6 December 2005

52. “Applications of Fluorescence Microscopy in Biomedical Research”, Biophysical Technology and Methods Frontiers Forum and 4th Chinese Biophysical Technologies Symposium. Tsing Hua University, China. 24 November 2005

51. “Nanotechnologies in biomedical applications”. Macau Biotechnology Research Institute, Macau, China, 22 November 2005

50. “Some applications in Multi-dimensional Microscopy”, 3rd Olympus Laser Confocal Microscope’s User Club Meeting, Shanghai, P.R. China, 19-20 September 2005

49. “Challenges in Liver Tissue Engineering?”, Symposium on "New Trends in Biomaterials-Tissue Engineering", Pan Pacific Hotel, Singapore, 9 July 2005

48. “Perfusion Culture of Liver Tissue Constructs”, ICMAT – ICAM 2005, Suntec Singapore International Convention and Exhibition Centre, Singapore, 4-8 July 2005

47. Focus on Microscopy 2005, Jena, Germany, 20-23 March 2005

46. “Engineering In Vitro Models for Metabolism Analysis of Drug Candidates”, Weekly Seminar for Division of Bioengineering, Nanyang Technological University, Singapore, 17 March 2005

45. “3-D Microscopy”, BMRC-EMBO Practical Course on Advanced Optical Methods in Cell and Developmental Biology, Biopolis, Singapore, 17-28 January 2005

44. “Overview of Stem Cell-Tissue Flagship programme”, SMA Symposium: Technical Parallel session for Computation and Systems Biology, Orchard Hotel, Singapore, 19-20 January 2005

43. “Tissue Engineering”, IBN Workshop for Science Teachers, Biopolis, Singapore, 3 December 2004

42. “Multidimensional Imaging Applications in Complex Tissue Engineering”, The Third International Conference on Structural Biology and Functional Genomics, National University of Singapore, Singapore, 2-4 December 2004

41. “In vitro Liver Tissue Models”, Medical Year 1 Foundation In Research Skills (FRS) AY2004/05, National University of Singapore, Singapore, 1-13 December 2004

40. “Development of Innovative Cell Technologies through engineering EC Microenvironment”, 2nd Shenzhen Forum in Biomedicine, Shenzhen, P.R. China, 3-7 November 2004

39. "Multidimensional Imaging Applications in Complex Tissue Engineering", Bioimaging Symposium: Emerging Technologies and Novel Techniques in Bioimaging, National University of Singapore, Singapore, 11 October 2004

38. "Tissue Engineering: An overview", Invitation lecture to graduate students in NUS, Faculty of Science (BL5204), Singapore, 7 October 2004

37. “Building Liver Tissue Structure and Functions from Scratch?” The First International SBE Conference on Bioengineering and Nanotechnology (ICBN), Biopolis, Singapore, 27-29 September 2004

36. “Precision engineering of Liver tissue Micro –Architecture AND Functions: Enabling Technologies”, Seminar at SBS-NTU, School of Biological Sciences, Nanyang Technological University, Singapore, 25 August 2004

35. “Precision engineering of Liver tissue Micro –Architecture AND Functions: Enabling Technologies”, 1st Nanoengineering and Nanoscience Congress, Singapore, 7-9 July 2004

34. BMRC Bioimaging Workshop, Biopolis, Singapore, 16 April 2004

33. “Re-look at Support for Heaptocytes Functions Ex Vivo”, Joint India-U.S. workshop on "Tissue Engineering and Stem Cell Technology", Trivandrum, India, 2-4 February 2004

32. Tissue Engineering Society International Annual Meeting, Orlando, Florida, USA, 10-13 December 2003

31. “Microenvironment for hepatocyte culture”, 2nd BMRC Symposium on Liver Research, Biopolis, Singapore, 28 November 2003

30. “Multidimensional Imaging of Cellular Dynamics in Engineered Microenvironment”, #217 XiangShan Forum: Bio- and Molecular Optical Imaging, HUST, Wuhan, P.R. China, 3-7 November 2003

29. “Confocal microscopy applications” AND “Live Imaging”, 2nd Olympus Laser Confocal Microscope’s User Club Meeting, Shanghai, P.R. China, 3-4 November 2003

28. “Kinectin functions: graduate students’ contributions”, The Graduate Students’ Society- Faculty of Medicine (GSS-FOM) 3rd Annual Scientific Conference “A Look Into The Future”, Singapore, April 2003

27. “Regenerative Medicine in Singapore” and “Liver Tissue Engineering”, kTi workshop and the second meeting of the Japanese Society of Regenerative Medicine”, Kobe, Japan, 10-12 March 2003

26. Beckman Coulter Cytomics Seminar, 21 February 2003, NUMI, NUS, Singapore.

25. “Multi-Disciplinary and Systematic Approaches to Reparative Medicine: Some Experiences From Liver Tissue Engineering”, Life Sciences in Singapore: Integrating Multidimensional Perspectives (4th Combined Scientific Meeting), Singapore, January 2003

24. “Our God and Life Sciences”, Singapore Bible College, Singapore, February 2003

23. “Liver Tissue Engineering” and “Nerve Tissue Engineering”, NUS-GTEC Pre-Conference Workshop on Engineering of Living Tissues, International Congress on Biological and Medical Engineering, Singapore, December 2002

22. “Liver and nerve tissue engineering",ICBME 2002. Singapore: BMES. (Keynote paper) Suntec City, Singapore, 6‑8 December 2002

21. “Issues and technologies leading to a new bio-artificial liver with micro-encapsulated hepatocytes”, Third Smith & Nephew International Symposium: Translating tissue engineering into products, Atlanta, USA, 13-16 October 2002

20. “Design, Implementation, & Analysis of Biological Experiments with High Resolution BioImaging Methods” & “Live Specimen Imaging Using Fluorescent Proteins, Quantum Dots, and Other Fluorescence Probes”, Summer School, Hsinghua University, China, 2002

19. “Environmental Control of Cellular Functions: A perspective in Liver Tissue Engineering”. Karolinska Institutet – NUS symposium on Tissue Engineering, Singapore, 2002

18. “Overview of the Mesenchymal and Adult Stem Cells”. Stem Cell Symposium plenary lecture, Singapore 2002

17. Singapore Society for Biochemistry & Molecular Biology: “Basic Science and Translational Research”, Singapore, 2002

16. “Career in Life Sciences”, Raffles Girl School, Singapore, 2002 & 2003

15. “Cell Biology and Technologies in Tissue Engineering”, Biology Department, Hong Kong University of Science and Technology, Hong Kong, December 2001

14. “Live Specimen Microenvironment for Multidimensional Imaging”, plenary lecture in Multidimensional Microscopy Conference (MDM2001), Melbourne, Australia, 25-28 November 2001

13. Confocal Microscopy and Digital Imaging Workshop, Fudan University, Department of Physiology, School of Life Sciences, Shanghai, China, 5-6 November 2001

12. Confocal Microscopy and Digital Imaging Workshop, Chinese Academy of Sciences, Institute of Genetics, Beijing, China, 1-2 November 2001

11. “Non-disruptive 3D cell culture and imaging for tissue engineering”, Symposium On Biomaterials & Tissue Engineering, International Conference on Materials For Advanced Technologies, Singapore, July 2001

10. “3D Microenvironment for Cellular Studies, Therapy and Tissue Engineering”, Pratt School of Engineering, Duke University, USA, February 2001

9. 34th Singapore-Malaysia Congress of Medicine/Combined Hospitals Medical & Dental Scientific Meeting: “Integrated approach to tissue regeneration”, Singapore, August 2000

8. Nanyang Technological University, Materials Engineering Academic Conference, plenary lecture, 2000: “Tissue Engineering: A multi-disciplinary endeavor, opportunities for engineers” Singapore, 2000

7. “Tissue Engineering Initiative in Singapore”, Biomedical Engineering Center, Industrial Technology Research Institute (ITRI), Hsinchu, Taiwan, 2000

6. “Liver Tissue Engineering With A Novel Cell Encapsulation System”, Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei, Taiwan, 2000

5. “Live Specimen Imaging: An Environmental Control”, Focus on Multidimensional Microscopy, KaoHsiung, Taiwan, 2000

4. “Tissue Engineering in Singapore”, Asian Society For Tissue Engineering Inaugural Meeting, Hiroshima, Japan, 2000

3. “Live Cell Imaging”, First Asia-Pacific Symposium on Confocal Microscopy & Related Technologies (SCMRT), Singapore, 1999

2. “Liver Tissue Engineering” Tissue Engineering Symposium, National University Hospital, 3rd Annual Scientific Meeting, Singapore, 1999

1. Microcopy Society of Singapore: “Confocal Microscopy: An Update”, Singapore, 1999

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