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Culture methods focusing on bile canalicular formation using primary human hepatocytes in a short time

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Abstract

The development of models for predicting hepatotoxicity is warranted, as the hepatotoxicity risk of 38–51% of compounds is undetectable in nonclinical studies. Cholestatic drug-induced liver injury (DILI) is a condition in which bile acids are abnormally excreted into the capillary bile canaliculi and are accumulated in hepatocytes, caused by the inhibition of bile salt export pump (BSEP), a transporter that is mainly associated with excretion of bile acids. Although laboratory animals are used as models, the use of human-derived cells is required owing to species differences. Unfortunately, primary human hepatocytes (PHHs) show rapid loss of function in culture and difficulties in forming bile canaliculi. Therefore, we aimed to develop an in vitro culture method for the efficient formation of bile canaliculi and for assessing the function of BSEP in PHHs. Here, PHHs were cultured from 1 h after thawing to day 2 with Z-VAD-FMK, a total caspase inhibitor, and RevitaCell™ supplement, an irreversible Rho-associated coiled-coil forming kinase (ROCK) inhibitor, in combination with RM-101. The PHHs formed bile canaliculi and showed BSEP function on day 6 of culture. Our findings suggest that cultured PHHs may improve the prediction accuracy of the risks of cholestatic DILI-contained toxicity on bile canaliculi.

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Acknowledgements

The authors thank Dr. Kousei Ito and Dr. Akinori Takemura (Chiba University, Japan) for providing the anti-BSEP polyclonal antibody.

Funding

This work was partly supported by Grants-in-Aid from the Japan Society for the Promotion of Science (Grants 20K16051 and 22K06773). Furthermore, support was also provided by the Research on Development of New Drugs from Japan Agency for Medical Research and Development (Grants 19be0304203h0003 and 20be0304203h0004).

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Authors and Affiliations

  1. Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-Dori, Mizuho-Ku, Nagoya, 467-8603, Japan

    Yoko Sakai, Masanari Matsumura, Takahiro Iwao & Tamihide Matsunaga

  2. Laboratory of Biological Chemistry, School of Pharmaceutical Sciences, Wakayama Medical University, Wakayama, Japan

    Yoko Sakai

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  1. Yoko Sakai
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  2. Masanari Matsumura
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  4. Tamihide Matsunaga
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Correspondence to Takahiro Iwao.

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Masanari Matsumura is a co-first author.

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Sakai, Y., Matsumura, M., Iwao, T. et al. Culture methods focusing on bile canalicular formation using primary human hepatocytes in a short time. In Vitro Cell.Dev.Biol.-Animal 59, 606–614 (2023). https://doi.org/10.1007/s11626-023-00805-y

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  • DOI: https://doi.org/10.1007/s11626-023-00805-y

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