Transgenic HepaRG cells expressing CYP2D6 as an improved model of primary human hepatocytes.

CYP2D6 and CYP3A4, which are members of the cytochrome P450 superfamily of metabolic enzymes, play major roles in the metabolism of commonly available drugs. CYP3A4 is involved in the metabolism of 50% of drugs on the market, whereas CYP2D6 is involved in the metabolism of 25% of them. CYP2D6 exhibits a high degree of polymorphic nature in the human population, causing individual differences in CYP2D6 expression and enzymatic activity. Therefore, accurate prediction of drug metabolism and toxicity require a human adult hepatocyte cell model that mimics individual responses in the average population. HepaRG cells, a human hepatocellular carcinoma cell line, is the only cell line that can differentiate into hepatocyte-like cells with high expression of CYP3A4 but poor expression of CYP2D6. To solve this problem, we developed transgenic HepaRG cell clones expressing either full-length or spliced CYP2D6 at various levels with an easy monitoring system for CYP2D6 expression in living cells under a fluorescent microscope. As CYP2D6 mRNA, protein, and fluorescence intensity were closely correlated among transgenic HepaRG clones, fluorescence levels will provide a simple tool for quality assurance of CYP2D6-expressing HepaRG cells. Thus, the package of transgenic HepaRG cell clones expressing CYP2D6 at various levels will provide an improved hepatocyte model that reflects the average or individual reactions in the human population for in vitro studies of drug metabolism and toxicity involving CYP2D6 and CYP3A4.

A two-dimensional multiwell cell culture method for the production of CYP3A4-expressing hepatocyte-like cells from HepaRG cells.

Cytochrome P450 enzymes (CYP) function in drug metabolism in the liver. To evaluate numerous drug candidates, a high-content screening (HCS) system with hepatocyte-like cells (HLCs) that can replace adult human hepatocytes is required. Human hepatocellular carcinoma HepaRG is the only cell line capable of providing HLCs with high CYP3A4 expression comparable to that in adult hepatocytes after cell differentiation. The aim of this study was to design an ideal multiwell culture system for HLCs using transgenic HepaRG cells expressing the EGFP coding an enhanced green fluorescent protein under CYP3A4 transcriptional regulation. HLCs were matured on five different types of 96-well black plates. Culturing HLCs on glass-bottom Optical CVG plates significantly promoted cell maturation and increased metabolic activity by twofold under two-dimensional (2D) culture conditions, and these features were enhanced by 2% collagen coating. Three plates for three-dimensional (3D) cell cultures with a gas-exchangeable fabric or dimethylpolysiloxane membrane bottom formed multiple round colonies, whereas they were ineffective for CYP3A4 expression. Under optimized conditions presented here, HLCs lost responsiveness to nuclear receptor-mediated transcriptional induction of CYP3A4, suggesting that CYP3A4 transcription has already been fully upregulated. Therefore, HepaRG-derived HLCs will provide an alternative to human hepatocytes with high levels of CYP3A4 enzyme activity even under 2D culture conditions. This will improve a variety of drug screening methods.