Bioengineering of Novel Organotypic 3D Human Liver/Hepatocyte Tissue Model for Drug Induced Liver Injury/Toxicity Studies

Development of a human primary cell-based 3D organotypic liver/hepatocyte tissue model with polarized hepatic morphology that maintains high level expression of major liver associated drug metabolizing enzymes is an attractive alternative platform to study acute and chronic liver toxicity and drug induced liver injury. In this study, we developed a human 3D hepatocyte tissue model using adult primary hepatocytes. To reconstruct the model, hepatocytes were seeded onto MatTek’s cell culture inserts and fed with specialized medium to form polarized and well differentiated hepatocyte tissue structure with defined apical and basolateral surfaces. Tissue morphology was characterized by histology, and albumin expression was evaluated by immunohistochemistry and ELISA. To monitor changes in gene expression levels for drug metabolizing enzymes associated with first pass metabolism, qPCR was performed at days 0, 10, 16 and 23 of the culture period. Utility of the tissue model for drug toxicity studies was demonstrated by dosing the reconstructed liver tissue with 100 µM of 5 model drugs (SN38, Bosentan, Diclofenac, Fialuridine, and Tolcapone) that are known to have adverse effects on liver in humans. Outcome measurements for liver toxicity include increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) release, two biomarkers with clinical relevance in liver functionality tests. Characterization of the tissue model showed 3D columnar hepatocyte tissue formation (histology), hexagonal cellular structure (topical view imaging), albumin production (immunohistochemistry), and albumin release to the basolateral and apical sides (ELISA). qPCR results demonstrated high level expression of enzymes involved in drug metabolism such as CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, and CYP4A11. Interestingly, repeated application of Fialuridine, a drug intended for hepatitis B treatment that was abruptly terminated due to induction of liver failure or causing of severe liver toxicity during clinical trials, showed an increase in ALT and AST levels in a time-dependent manner at days 5 and 7, which is indicative of drug induced liver injury (DILI). The positive control, SN38, a metabolite of the cancer drug Irinotecan, also showed an increase in ALT and AST levels. Development of this novel 3D human liver tissue model using primary adult hepatocytes creates an opportunity to study liver physiology in an in vitro tissue platform. The developed liver model can also play a key role in screening potential liver toxicity of candidates that are in the drug development pipeline.  Such a model will allow formulation scientists to identify adverse effects of therapeutic candidates early in the drug development process. This model will also  reduce animal use for experimentation.

EpiLiver, liver, toxicity, hepatocytes, albumin, SN38, Irinotecan, Bosentan, Diclofenac, Fialuridine, Tolcapone, alanine amino transferase, aspartate amino transferase, drug metabolizing enzymes first pass metabolism, cytochrome P450, CYP450, CYP1A1, CYP1A2, CYP26A1, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2S1, CYP3A4, CYP3A5, CYP3A7, CYP4A11, CYP4F11, CYP4F3, CYP7A1, CYP7B1, CYP8B1, GSTA1, GSTA3, GSTA4, SULT1A1, SULT1B1, UGT1A1, UGT1A4, UGT1A9, UGT2A3, UGT2B10, UGT2B17, UGT2B28, UGT2B4, UGT2B7, UGT3A1

SN38, Irinotecan, Bosentan, Diclofenac, Fialuridine, Tolcapone