Abstract
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.
Highlights
Drug-Induced Liver Injury (DILI) is the major severe adverse drug reaction of marketed drugs, with severe health consequences for the patients and financial loss for the pharmaceutical industry due to market withdrawal [1]
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
Primary hepatocytes cultured as a monolayer are commonly used, their application is restricted to the assessment of acute toxicity due to limited viability, altered polarity, rapid loss of liver-specific gene expression and rapidly decreasing functionality [2,3]
Summary
Drug-Induced Liver Injury (DILI) is the major severe adverse drug reaction of marketed drugs, with severe health consequences for the patients and financial loss for the pharmaceutical industry due to market withdrawal [1]. The rapidly altered gene expression of PCLS upon cultivation restricts their use to short-term applications and does not allow the investigation of drug-induced subchronic toxicity [5,11]. In addition to their restricted metabolic activity during prolonged cultivation, a major limitation of PCLS is the requirement of complex and sophisticated dynamic culture conditions, preventing high-throughput screening applications [5]. 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 Their use as a reliable predictive in vitro model for drug-induced liver injury is limited. Several liver-specific pathways remain to be characterized in such models
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