Optimization of maturity levels of human hepatic cell lines in tissue culture

It is generally thought that proliferation and full maturation of hepatocytes are mutually exclusive. For this reason human hepatic cell lines unlimitedly proliferate but with reduced maturity levels under standard culture conditions.We set out to optimize culture conditions for the hepatic cell lines Huh–7 and HepG2 towards less proliferation and more maturity based on expression levels of maturity markers like metabolic enzymes, protein members of tight and adherens junctions etc.–in comparison to levels reached in freshly isolated primary human hepatocytes.Huh–7 cells cultured with DMSO lead to cells of advanced maturity, which can be used for production of the chimeric JFH–1 HCV laboratory strain in DMEM-based standard medium [1]–a cultivation protocol we used as baseline. So far, in a first series of experiments, cells were cultured in HBM (Lonza) supplemented with Hydrocortisone, Transferrin, Insulin, Oncostatin M, Dexamethasone and human serum in adherent and suspension culture for two weeks with and without DMSO. Expression analyses by RT-qPCR revealed stronger expression of liver enriched transcription factors in adherent conditions with DMSO than in suspension. In contrast, expression of gap- and tight-junction and metabolic transcripts were upregulated in suspension, accompanied by strong proliferation arrest. Furthermore, use of human serum as replacement for FCS and no DMSO supplementation in the second week of culture enhanced expression of maturity markers in particular in suspension culture. Further experiments dealing with different and more combinations of media supplementations are currently tested and data will be presented.Once a satisfying optimized maturation protocol for Huh–7 and HepG2 cells is found, the ultimate aim of the studies would be to screen a variety of commonly used hepatic cell lines for i) their support for replication of HCV and HBV and ii) their suitability for in vitro drug testings under maturation conditions.

Literatur: [1] Production of Infectious Hepatitis C Virus by Well-Differentiated, Growth-Arrested Human Hepatoma-Derived Cells Bruno Sainz Jr., and Francis V. Chisari Journal of Virology, October 2006, p. 10253-10257, Vol. 80, No. 20

Maturation - liver cell lines - tissue culture