Functional Proliferating Human Hepatocytes: In Vitro Hepatocyte Model for Drug Metabolism, Excretion, and Toxicity

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.

Three-Dimensional Liver Culture Systems to Maintain Primary Hepatic Properties for Toxicological Analysis In Vitro

“…The expanded cells can be converted back into mature hepatocytes at any desired time-point using specialized hepatocyte maturation media [ 47 ]. Others keep pHHs proliferating by using a complex medium composition and hypoxic conditions [ 48 ].…”

“…Gene-expression profiling showed that spheroids of primary-like hepatocytes clustered with pHHs and differentiated HepaRG cells, but not with HepG2 cells, indeed indicating a mature phenotype [ 106 ]. And a third study showed that spheroids of so called ProliHHs were comparable to pHHs regarding albumin expression and secretion, but expression and activity levels of CYP1A2, CYP2B6 and CYP3A4, as well as BSEP and MRP2 expression levels were considerably lower than in pHHs [ 48 ].…”

Current strategies with implementation of three-dimensional cell culture: the challenge of quantification

“…A stepping stone between 2D and 3D, sandwich cultures have been established for three decades [15] whereby a 2D cell layer is placed between thin layers comprising ECM proteins. They provided an early demonstration of the benefits of more complex culture systems, for example, hepatocyte activity and function were much more analogous to hepatocytes in vivo [16][17][18][19].…”

FEK self‐assembled peptide hydrogels facilitate primary hepatocytes culture and pharmacokinetics screening

“…Next, supernatants were collected followed by measurement of metabolized compounds via LC–MS/MS. Corresponding substrates of metabolic enzymes used for analysis were Cytochrome P450 3A4 (Cyp3a4), testosterone; Cytochrome P450 1A2 (Cyp1a2), phenacetin; Cytochrome P450 2B6 (Cyp2b6), bupropion; Cytochrome P450 2C9 (Cyp2c9), diclofenac; Cytochrome P450 2E1 (abbreviated Cyp2e1), chlorzoxazone 32,33 …”