Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (S.Q., Z.W., C.M., Z.P., G.P.); University of Chinese Academy of Sciences, Beijing, China (S.Q., Z.W., C.M., Z.P., G.P.); Shanghai Hexaell Biotech Co., Ltd, Shanghai, China (S.F., E.T.); Nanjing University of Chinese Medicine, Nanjing, China (Z.W.); and Nanjing Tech University, Nanjing, China (T.H.).
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China (S.Q., Z.W., C.M., Z.P., G.P.); University of Chinese Academy of Sciences, Beijing, China (S.Q., Z.W., C.M., Z.P., G.P.); Shanghai Hexaell Biotech Co., Ltd, Shanghai, China (S.F., E.T.); Nanjing University of Chinese Medicine, Nanjing, China (Z.W.); and Nanjing Tech University, Nanjing, China (T.H.)
Drug Metab Dispos. 2021 Apr;49(4):305-313. doi: 10.1124/dmd.120.000275. Epub 2021 Feb 1.
To develop a functional alternative hepatocyte model for primary human hepatocytes (PHHs) with proliferative property, essential drug metabolic, and transporter functions, proliferating human hepatocytes (ProliHHs) expanded from PHHs were fully characterized in vitro. Herein, ProliHHs generated from multiple PHHs donors could be expanded more than 200-fold within four passages and maintained their metabolic or transporter capacities partially. Furthermore, ProliHHs were able to regain the mature hepatic property after three-dimensional (3D) culture. Particularly, the downregulated mRNA expression and function of three major cytochrome P450 (P450) enzymes (CYP1A2, CYP2B6, and CYP3A4) in the proliferating process (ProliHHs-P) could be recovered by 3D culture. The metabolic variabilities across different PHHs donors could be inherited to their matured ProliHHs (ProliHHs-M). The intrinsic clearances of seven major P450 enzymes in ProliHHs-M correlated well (r = 0.87) with those in PHHs. Also, bile canaliculi structures could be observed in sandwich-cultured ProliHHs (SC-ProliHHs), and the biliary excretion index of four probe compounds [cholyl-lys-fluorescein, 5-(and-6)-carboxy-2', 7'-dichlorofluorescein diacetate (CDF), deuterium-labeled sodium taurocholate acid, and rosuvastatin] in SC-ProliHHs (>10%) were close to sandwich-cultured PHHs. More importantly, both ProliHHs-P and ProliHHs-M could be used to evaluate hepatotoxicity. Therefore, these findings demonstrated that the 3D and sandwich culture system could be used to recover the metabolic and transporter functions in ProliHHs for clearance prediction and cholestasis risk assessment, respectively. Together, ProliHHs could be a promising substitute for PHHs in drug metabolism, transport, and hepatotoxicity screening. SIGNIFICANCE STATEMENT: This report describes the study of drug metabolic capacities, efflux transporter functions, and toxicity assessments of proliferating human hepatocytes (ProliHHs). The metabolic variability in different primary human hepatocyte donors could be inherited by their matured ProliHHs derivatives. Also, ProliHHs could form canalicular networks in sandwich culture and display biliary excretion capacities. More importantly, both the proliferative and maturation statuses of ProliHHs could be used to evaluate hepatotoxicity. Together, ProliHHs were feasible to support drug candidate screening in hepatic metabolism, disposition, and toxicity.
为了开发具有增殖特性、必需药物代谢和转运功能的功能性替代原代人肝细胞（PHH）的人肝细胞模型，我们对源自 PHH 的增殖人肝细胞（ProliHH）进行了全面的体外表征。在此，源自多个 PHH 供体的 ProliHH 可在 4 个传代内扩增超过 200 倍，并部分保持其代谢或转运能力。此外，ProliHH 在三维（3D）培养后能够恢复成熟的肝特性。特别是，在增殖过程中（ProliHH-P）三种主要细胞色素 P450（CYP）酶（CYP1A2、CYP2B6 和 CYP3A4）的下调 mRNA 表达和功能可通过 3D 培养恢复。不同 PHH 供体之间的代谢变异性可遗传至成熟的 ProliHH（ProliHH-M）。ProliHH-M 中 7 种主要 CYP 酶的内在清除率与 PHH 中的清除率相关性良好（r=0.87）。此外，在三明治培养的 ProliHH（SC-ProliHH）中可以观察到胆小管结构，并且四种探针化合物[胆酰-赖氨酸-荧光素、5-（和 6）-羧基-2'，7'-二氯荧光素二乙酸酯（CDF）、氘标记的牛磺胆酸钠和瑞舒伐他汀]在 SC-ProliHH 中的胆汁排泄指数（>10%）接近三明治培养的 PHH。更重要的是，ProliHH-P 和 ProliHH-M 均可用于评估细胞毒性。因此，这些发现表明，3D 和三明治培养系统可用于恢复 ProliHH 中的代谢和转运功能，分别用于清除预测和胆汁淤积风险评估。总之，ProliHH 可以作为 PHH 在药物代谢、转运和细胞毒性筛选中的有前途的替代品。
本报告描述了对增殖人肝细胞（ProliHH）的药物代谢能力、外排转运功能和毒性评估的研究。不同原代人肝细胞供体的代谢变异性可通过其成熟的 ProliHH 衍生物遗传。此外，ProliHH 可在三明治培养中形成胆小管网络并显示胆汁排泄能力。更重要的是，ProliHH 的增殖和成熟状态均可用于评估细胞毒性。总之，ProliHH 可用于支持候选药物在肝脏代谢、处置和毒性方面的筛选。
