Generation of expanded primary hepatocytes for cell based toxicity and metabolism screenings

Primary human hepatocytes are routinely used to evaluate biotransformation, enzyme induction/inhibition and toxicity of novel drug candidates. However, their use is compromised by the limited availability of human liver tissue as well as a rapid dedifferentiation and lack of proliferation in vitro. To address this, we recently introduced a technique which causes primary hepatocytes to proliferate for up to 40 population doublings while maintaining a mature phenotype (upcyte® hepatocytes).

In the current study, we evaluated the suitability of upcyte® hepatocytes as a hepatic model by analyzing certain hepatic markers such as production of human serum albumin (HSA) and their metabolic activity and their use for preclinical cell-based assays. Expanded upcyte® cells revealed high basal activities of phase I enzymes which were inducible by prototypical inducers like rifampicin (CYP3A4, CYP2B6, CYP2C8, CYP2C9). We further detected primary-like activity of several phase II enzymes such as e.g. UDP-glucuronosyltransferase & glutathione S-transferase.

We have tested the cells in several applications such as cytotoxicity, genotoxicity, viral infection, 3D cultures and their use in clearance prediction. upcyte® hepatocytes predicted acute and long-term hepatotoxicity of established model compounds like acetaminophen, troglitazone and cyclosporine A as determined by multiparametric HCS analysis. Affected parameters included overall viability, apoptosis, mitochondrial membrane potential, oxidative stress, lipid accumulation and intracellular Ca2 levels. Proliferation of upcyte® cells enabled us to perform genotoxicity screenings. Here, we observed a significant increase in micronuclei formation in response to genotoxic agents like mitomycin C and cyclophosphamide.

We next determined whether upcyte® hepatocytes respond to infection with hepatitis C virus (HCV). Replication and infectivity of HCV was observed as indicated by intracellular NS5A levels and focus-forming assay. Co-culture of upcyte® hepatocytes with additional liver cell types (liver sinusoidal endothelial cells, mesenchymal stem cells, hepatic stellate cells) on a Matrigel® matrix induced formation of organoids displaying typical functional characteristics of liver parenchyma including elevated phase I activities and expression of hepatic markers.

Taken together, our results suggest that upcyte® hepatocytes display many primary-like features during pre-clinical in vitro assays. Given the additional benefit of virtually unlimited cell access, upcyte® hepatocytes represent a promising hepatic model in biomedical research and drug development.