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Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

ABSTRACT: Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain ?2, ?3, ?4, ?1, ?3, ?1, and ?2 were detected in both isolated human hepatocytes and liver tissue. No ?1 and ?5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human hepatocytes, and that recombinant laminin is a promising xeno-free and chemical defined strategy for preservation of hepatocyte specific function in vitro.

SUBMITTER: Watanabe M

PROVIDER: S-EPMC5012698 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

Watanabe Masaaki M Zemack Helen H Johansson Helene H Hagbard Louise L Jorns Carl C Li Meng M Ellis Ewa E

PloS one 20160906 9

Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions o ...[more]

PMID: 27598296

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Project description:BackgroundIn recent years, differentiation of human induced pluripotent stem cells (hiPSCs) into brain-specific microvascular endothelial cells (iBMECs) has frequently been used to model the blood-brain barrier (BBB). However, there are limitations in the use of iBMECs for in vitro studies, such as transendothelial electrical resistance (TEER) instability, weak junctional expression of VE-cadherin, and lack of proper fluid shear stress response. In vivo, the basement membrane (BM) composition of the BBB evolves throughout development, and laminins become the dominant component of the mature vascular BM. However, laminin isoforms of the endothelial BM have not been used for culture of differentiated iBMECs. The main goal of this study is to investigate the effect of different laminin isoforms of the endothelial BM on iBMEC functionality and to determine whether better recapitulation of the physiological BM in vitro can address the aforementioned limitations of iBMECs.MethodsUsing a previously reported method, hiPSCs were differentiated into iBMECs. The influence of main laminins of the endothelial BM, LN 411 and LN 511, on iBMEC functionality was studied and compared to a collagen IV and fibronectin mixture (CN IV-FN). Quantitative RT-PCR, immunocytochemistry, and TEER measurement were utilized to assess gene and protein expression and barrier properties of iBMECs on different extracellular matrices. Single-channel microfluidic devices were used to study the effect of shear stress on iBMECs.ResultsLN 511, but not LN 411, improved iBMEC barrier properties and resulted in more sustained TEER stability. Immunocytochemistry showed improved junctional protein expression compared to iBMECs cultured on CN IV-FN. iBMECs cultured on LN 511 showed a reduction of stress fibers, indicating resting endothelial phenotype, whereas gene expression analysis revealed upregulation of multiple genes involved in endothelial activation in iBMECs on CN IV-FN. Finally, culturing iBMECs on LN 511 enhanced physiological responses to shear stress, including morphological changes and enhanced junctional protein association.ConclusionLN 511 improves the functionality and long-term barrier stability of iBMECs. Our findings suggest that incorporation of physiologically relevant LN 511 in iBMEC culture would be beneficial for disease modeling applications and BBB-on-a-chip platforms that accommodate fluid flow.

Dataset Information

Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

Publications

Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

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