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 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.
Highlights
During the cell isolating process, hepatocytes are separated from each other and deprived of their original environment [1]
Isolated primary human hepatocytes were cultured on different laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521, Engelbreth-Holm-Swarm murine sarcoma (EHS), or Collagen pre-coated plates, and were evaluated morphologically
Hepatocytes cultured on EHS migrated within a few days and formed threedimensional cell clusters while hepatocytes cultured on laminin or Collagen showed characteristic, hexagonal shape in a flat cell monolayer for up to six days (Fig 2)
Summary
During the cell isolating process, hepatocytes are separated from each other and deprived of their original environment [1]. The disruption of cell-to-cell and cell-to-matrix contacts leads to subsequent hepatocyte dysfunction and/or apoptosis, and the isolated hepatocytes lose their specific functions if the cells are not anchored and cultured under appropriate conditions [2,3,4,5]. The extracellular matrix contains different specific proteins, such as laminins, type IV collagens, perlecan, and nidogens, forming complex networks that strongly influence the behavior and functions of the associated cells [6]. To maintain specific functions in a culture system, isolated hepatocytes need to be cultured with an extracellular matrix component, usually MatrigelTM or collagen. Clinical application under good manufacturing practice (GMP) protocols is difficult due to their animal origin. A robust and new strategy for culturing isolated hepatocytes under xeno-free and chemical defined conditions is important for future use of primary and/or stem cell derived hepatocytes in GMP settings
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call