No abstract

To develop a functional alternative hepatocyte model for primary human hepatocytes (PHH) with proliferative property, essential drug metabolic and transporter functions, proliferating human hepatocytes (ProliHH) expanded from PHH were fully characterized in vitro. Herein, ProliHH generated from multiple PHH donors could be expanded more than 200 folds within four passages, and maintained their metabolic or transporter capacities partially. Further, ProliHH was able to regain of the mature hepatic property after three-dimensional (3D) culture. Particularly, the downregulated mRNA expression and function of three major CYP450 enzymes (CYP1A2, CYP2B6, and CYP3A4) in the proliferating process (ProliHH-P) could be recovered by 3D culture. The metabolic variabilities across different PHH donors could be inherited to their matured ProliHH (ProliHH-M). The intrinsic clearances (CL int ) of seven major CYP450 enzymes in ProliHH-M were correlated well (r = 0.87) with that in PHH. Also, bile canaliculi structures could be observed in sandwich cultured ProliHH (SC-ProliHH), and the biliary excretion index (BEI) of four probe compounds (CLF, CDF, d8-TCA and rosuvastatin) in SC-ProliHH (>10%) were close to SC-PHH. More importantly, both ProliHH-P and ProliHH-M could be used to evaluate hepatotoxicity. Therefore, these findings demonstrated that the 3D and sandwich culture system could be utilized to recover the metabolic and transporter functions in ProliHH for clearance prediction and cholestasis risk assessment, respectively. Together, ProliHH could be a promising substitute for PHH in drug metabolism, transport and hepatotoxicity screening.