Drug metabolic activity is a critical cell-intrinsic determinant for selection of hepatocytes during long-term culture

Abstract

The liver plays many important roles in homeostasis, including drug detoxification, metabolism, and bile production. Hepatocytes, which are the main constituent cells of the liver, play an important role in the pathogenesis of liver diseases, identification of candidate compounds in drug discovery research, pharmacokinetic studies, and toxicity evaluation. Human hepatocytes are, however, difficult to grow in normal in vitro culture systems, making it difficult to secure cell numbers. As an alternative, evaluation systems using animal models and hepatocellular carcinoma cells have been established, but interspecies and interracial differences and low hepatic function have been pointed out as problems. Therefore, there is still a need for a highly stable method to prepare human hepatocytes with sufficient functionality. In this study, we aimed to establish an in vitro long-term culture system that enables stable proliferation and maintenance of the functionality of human hepatocytes to stably supply human hepatocytes. In the established culture system, the stable proliferation of human hepatocytes was achieved by co-culturing hepatocytes with mouse fetal fibroblasts to dedifferentiate them into hepatic progenitor-like cells. Furthermore, we succeeded in purifying human hepatocytes by puromycin with a rapid cytocidal effect and proliferating them to over 30 population doublings for more than 200 days. Hepatocytes with high expression of cytochrome P450 genes survived after exposure to cytotoxic antibiotics because of enhanced drug-metabolizing activity. These results show that the above culture system enables simple and efficient hepatocyte proliferation, and is considered to be an effective method for stable supply of hepatocytes and significant cost reduction in drug discovery research.

Drug metabolic activity as a selection factor for pluripotent stem cell-derived hepatic progenitor cells

“…Resistance to puromycin may be due to the high metabolic activities of CYP3A4. This metabolism of puromycin by CYP3A4 can indeed be predicted by the computer algorithm "admetSAR" and previous reports 23,37,38 .…”

“…Puromycin equalized hepatocytes with diverse functions such as drug metabolism after the in vitro propagation. The stable proliferative capacity of primary hepatocytes and pluripotent stem cell-derived hepatocytes requires WNT, R-sponsin1, and feeder cells 23,[33][34][35][36] . Resistance to puromycin may be due to the high metabolic activities of CYP3A4.…”

“…In previous studies, we achieved efficient proliferation by co-culturing irrMEFs with hepatocytes derived from patients with liver disease 23 . When human ESC-hepatocytes were passaged and co-cultured with irrMEFs, the cells formed colonies.…”

“…Recently, it has been reported that puromycin treatment is effective in selecting proliferative hepatocytes from the liver of drug-induced liver injury (DILI) patients 23 . Proliferative hepatocytes selected by this method exhibit stable and high proliferative potential in Wnt-containing media and MEF feeders.…”