Journal of Tissue Science and Engineering

Abstract Background: Primary hepatocytes rapidly lose their polarized morphology and the expression of important liverspecific metabolic enzymes, receptors and transport proteins under normal two-dimensional (2D) culture conditions. Thus, their use as a reliable predictive in vitro model for drug-induced liver injury is limited. Three-dimensional (3D) liver micro tissue culture systems have become an increasingly attractive alternative for the evaluation of druginduced liver toxicity. However, several liver-specific pathways remain to be characterized in such models. Methods and principal findings: In the present work, we compared the expression of several genes with a role in the anti-oxidant cell defense and glucocorticoid pathways in rat H4IIE hepatoma cells, primary rat hepatocytes, 2D-hepatocyte sandwich culture, and a multi-cell type micro tissue model comprising primary rat hepatocytes in coculture with liver-derived nonparenchymal cells and macrophage (Kupffer cells). Gene expression was studied for up to 25 days in culture. High expression levels of the Nrf2-dependent genes NQO1, ABCC3 and GST2A were detected in H4IIE cells and in 3D-liver microtissues, in contrast to 2D-hepatocytes where a rapid decline of these genes was observed. The glucocorticoid-dependent genes ORM1, G6PC, PCK1 and HSD11B1 were highly expressed up to 25 days of cultivation in 3D-liver microtissues, but they showed very low or background expression in H4IIE and 2D-hepatocyte models. Conclusions: The tested 3D-multi-cell type liver micro tissue represents a stable and functionally active model system, with sustained expression for more than three weeks of cultivation of important metabolic proteins regulated by the glucocorticoid and anti-oxidant cell defense pathway.