Human liver cell cultivation for long-term in vitro toxicity studies in a miniaturized multi-compartment 3D bioreactor system

Classical model systems of hepatotoxicity have profound limitations in adequately assessing drug toxicity: Results from animal experiments cannot necessarily be extrapolated to humans; primary human hepatocytes lose their specific metabolic functions within short time in conventional 2D cell culture. Based on a liver cell bioreactor for clinical use in an extracorporeal liver support system employing hollow fiber perfusion and internal oxygenation a down-scaled system (0.5ml cell volume) for in vitro studies was recently developed. This study aims to investigate the miniaturized bioreactor’s capacity for long-term maintenance of primary human hepatocytes and their specific metabolic functions in respect of feasibility for pharmacological studies.The study comprises culture of liver cells in the bioreactor system for up to 3 weeks with regular measurement of different metabolic parameters. Cytochrome P450 (CYP) activities, including 2D6, 1A2, 2B6, 2C9 and 3A4, and urea production as indicators for specific hepatic metabolism were shown to be maintained for at least 10 days of culture. Aspartate transaminase and glutamate dehydrogenase release, measured to assess cellular injury, peaked around day 2 to 3 and thereafter stabilized on basal levels. Immunochemically assessed expression patterns of cytokeratin (CK) CK19 as biliary marker, CK18 as hepatocyte/biliary marker and the mesenchymal marker vimentin confirmed the preservation of cellular identity. 2*107 cells were sufficient to achieve tissue like densities. Moreover, an initial pharmacological study was performed with diclofenac, a model drug for idiosyncratic hepatotoxicity. Here, the time course for metabolite formation was analyzed for two diclofenac concentrations.The results indicate that the miniaturized bioreactor system facilitates prolonged hepatocyte culture while preserving specific metabolism. In addition the feasibility of the system for in vitro drug metabolism testing could be demonstrated.

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Literatur:

Gerlach JC, Mutig K, Sauer IM, Schrade P, Efimova E, Mieder T, Naumann G, Grunwald A, Pless G, Mas A, Bachmann S, Neuhaus P, Zeilinger K.Use of primary human liver cells originating from discarded grafts in a bioreactor for liver support therapy and the prospects of culturing adult liver stem cells in bioreactors: a morphologic study.Transplantation. 2003 Sep 15;76(5):781-6.

3D culture - bioreactors - human hepatocytes - in vitro model - liver cell culture - pharmacological testing