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

• Published in: PloS one 2016-09, Vol.11 (9), p.e0161383-e0161383
• Main Authors: Watanabe, Masaaki, Zemack, Helen, Johansson, Helene, Hagbard, Louise, Jorns, Carl, Li, Meng, Ellis, Ewa
• Format: Article
• Language: English
• Subjects: Bile acids; Biology and Life Sciences; Catheters; Cell culture; Cell Differentiation - drug effects; Cell Survival - drug effects; Collagen; Collagen (type IV); Consent; Culture Media - chemistry; Cytochrome; Cytochrome P450; Deoxyribonucleic acid; DNA; Extracellular matrix; Gene expression; Good Manufacturing Practice; Health aspects; Hepatocyte nuclear factor 4; Hepatocytes; Hepatocytes - cytology; Hepatocytes - drug effects; Hepatocytes - metabolism; Humans; Isoforms; Laminin; Laminin - chemistry; Laminin - genetics; Laminin - pharmacology; Liver; Liver - cytology; Liver - drug effects; Liver Regeneration - drug effects; Liver Regeneration - genetics; Liver transplants; Medicin och hälsovetenskap; Medicine and Health Sciences; Metabolism; Multidrug resistance; Phosphates; Physiological aspects; Preservation; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - pharmacology; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - pharmacology; Rodents; Sarcoma; Science; Stem cells; Surgery; Tissue engineering
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0161383

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 α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 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.