A human hepatic 3D-cell culture model for basic research, preclinical drug metabolism and toxicity testing

Introduction: Drug-induced toxicity is a main cause of acute liver failure. To test drug metabolism and toxicity and to reduce adverse drug reactions and animal experimentation, highly predictive human hepatic in vitro models are strong requested. We have established a three-dimensional (3D) liver cell model for drug discovery, maintaining liver-specific functions in the longer term.

Methods: HepaRG, a human progenitor cell line, which is able to differentiate into hepatocyte-like cells, were cultured in a porous polystyrene (PS) matrix to reach more physiological culture conditions than in a 2D cell model. Differentiation of HepaRG cells was induced by dimethyl sulfoxide. HepaRG cell morphology and settling were investigated using haematoxylin staining, formazan formation, light microscopy (LM). Cell proliferation (MTT assay) and cell cytotoxicity (LDH assay) were determined. Thereafter, CYP450 expression was analyzed by qRT-PCR and activity by fluorescence based assays. Albumin synthesis was measured over time. The effect of Acetaminophen (AAP) on the metabolic activity and hepatoxicity was determined.

Results: Cell staining and formazan formation revealed an uniform distribution of HepaRG cells in the PS matrix. Analysis of cell viability, cytotoxicity and cell proliferation proved that PS scaffolds are well tolerated by HepaRG cells. Differentiation of HepaRG cells within PS scaffolds resulted in significantly higher mRNA expression and activity levels of important biotransformation phase I CYP450 genes compared to the conventional 2D cell model. AAP showed a dose dependent effect on the metabolic activity and toxicity in differentiated 3D liver cell model.

Discussion: The differentiated 3D HepaRG cell model is a promising in vitro system for human liver cell-based test applications and might help to support the reduction of test animals. It has clear benefits above the 2D model and better represents the in vivo situation.

3D cell model - HepaRG - acetaminophen - scaffold