Abstract
Conventional in vitro human hepatic models for drug testing are based on the use of standard cell lines derived from hepatomas or primary human hepatocytes (PHHs). Limited availability, interdonor functional variability and early phenotypic alterations in PHHs restrict their use, whilst standard cell lines such as HepG2 lack a substantial and variable set of liver‐specific functions such as CYP450 activity. Alternatives include the HepG2‐derivative C3A cells selected as a more differentiated and metabolically active hepatic phenotype. Human HepaRG cells are an alternative organotypic co‐culture model of hepatocytes and cholangiocytes reported to maintain in vivo‐like liver‐specific functions, including intact Phase I–III drug metabolism. In this study, we compared C3A and human HepaRG cells using phenotypic profiling, CYP450 activity and drug metabolism parameters to assess their value as hepatic models for pre‐clinical drug testing or therapeutics. Compared with C3As, HepaRG co‐cultures exhibit a more organotypic phenotype, including evidence of hepatic polarity with the strong expression of CYP3A4, the major isoform involved in the metabolism of over 60% of marketed drugs. Significantly greater CYP450 activity and expression of CYP1A2, CYP2E1 and CYP3A4 genes in HepaRG cells (comparable with that of human liver tissue) was demonstrated. Moreover, HepaRG cells also preferentially expressed the hepatic integrin α5β1 – an important modulator of cell behaviour including growth and survival, differentiation and polarity. Drug metabolite profiling of phenacetin (CYP1A2) and testosterone (CYP3A4) using LC‐MS/MS and HPLC, respectively, revealed that HepaRGs had more intact (Phase I–II) metabolism profile. Thus, HepaRG cells significantly outperform C3A cells for the potential pharmaceutical and therapeutic applications.
Highlights
The human HepaRG hepatic cell line has emerged as a potential surrogate to Primary human hepatocytes (PHHs) for preclinical hepatotoxicity assays[14]
Phenotypic profiling of HepaRG and HepG2/C3A cells HepaRG cells formed terminally-differentiated in vivo-like hepatic cords and cholangiocytelike cells with functional polarity, as evidenced by punctate staining of F-actin bands indicative of bile-canalicular structures [phalloidin-staning, fig. 1c]; these could be observed in C3A cells, with less pronounced staining
Biotransformation potential CYP450 enzyme activity and specificity HepaRG CYP1A2 and CYP3A4 activity was significantly higher than that measured in HepG2/C3A cells [grey bars; p
Summary
The human HepaRG hepatic cell line has emerged as a potential surrogate to PHHs for preclinical hepatotoxicity assays[14]. HepaRGs are a unique (intrinsic), terminally-differentiated co-culture of hepatocyte- and cholangiocyte-like cells containing many functional and phenotypic similarities with PHH15. These cells were procured from a young adult female with hepatocarcinoma[16]. HepaRG cells retain some of the major CYP450 pathways and Phase II, enzymes - as well as production of glucose/glycogen and urea[15-17] These cells show functional polarity, a hallmark of in vivo hepatocyte organisation[15], with intact Phase III drug transporters. These properties are generally not evident in ‘standard’ human hepatic cell lines monocultures. We aimed to compare phenotypic and metabolic parameters, including CYP450 activity and metabolism between HepG2/C3A and human HepaRG cells, to assess their value as suitable hepatic models for pre-clinical drug testing and therapeutics
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