Culture density contributes to hepatic functions of fresh human hepatocytes isolated from chimeric mice with humanized livers: Novel, long-term, functional two-dimensional in vitro tool for developing new drugs

PLoS One. 2020 Sep 11;15(9):e0237809. doi: 10.1371/journal.pone.0237809. eCollection 2020.

Abstract

Chimeric mice with humanized livers are considered a useful animal model for predicting human (h-) drug metabolism and toxicity. In this study, the characteristics of fresh h-hepatocytes (cFHHs, PXB-cells®) isolated from chimeric mice (PXB-mice®) were evaluated in vitro to confirm their utility for drug development. cFHHs cultured at high density (2.13 × 105 cells/cm2) displayed stable production of h-albumin and cytochrome P450 (CYP) 3A activities for at least 21 days. The mRNA expression levels of 10 of 13 CYP, UDP-glucuronosyltransferase (UGT), and transporters were maintained at >10% of the levels of freshly isolated cFHHs after 21 days. From 1 week, many bile canaliculi were observed between cFHHs, and the accumulation of the multidrug resistance-associated protein and bile salt export pump substrates in these bile canaliculi was clearly inhibited by cyclosporin A. Microarray analysis of cFHHs cultured at high density and at low density (0.53 × 105 cells/cm2) revealed that high density culture maintained high expressions of some transcription factors (HNF4α, PXR, and FXR) perhaps involved in the high CYP, UGT and transporter gene expressions of cFHHs. These results strongly suggest that cFHHs could be a novel in vitro tool for drug development studies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bile Canaliculi / cytology
  • Bile Canaliculi / drug effects*
  • Bile Canaliculi / metabolism
  • Cells, Cultured
  • Child
  • Child, Preschool
  • Cyclosporine / pharmacology
  • Cytochrome P-450 CYP3A / genetics
  • Cytochrome P-450 CYP3A / metabolism
  • Drug Development / methods*
  • Female
  • Glucuronosyltransferase / genetics
  • Glucuronosyltransferase / metabolism
  • Hepatocytes / cytology
  • Hepatocytes / drug effects*
  • Hepatocytes / metabolism
  • Humans
  • Male
  • Mice
  • Mice, SCID
  • Primary Cell Culture / methods*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transplantation Chimera*

Substances

  • Transcription Factors
  • Cyclosporine
  • Cytochrome P-450 CYP3A
  • Glucuronosyltransferase

Grants and funding

This research was supported by the Research on Development of New Drugs from Japan’s Agency for Medical Research and Development, AMED. PhoenixBio Co., Ltd. also provided support for this study in the form of salaries for: CY, YI, AY, YY, YK, YO, YK, YI, CT. The specific roles of these authors are articulated in the ‘author contributions’ section. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.