Considerations for developing CYP induction assays in hepatocytes: Insights from a multilaboratory study
Cytochrome P450 (CYP) induction studies using primary human hepatocytes (PHH) were conducted across seven laboratories. Standard operating procedures (SOPs) were developed and distributed, ensuring all laboratories used PHH from the same donor and CYP inducers prepared at a single location. In each laboratory, PHH was seeded, cultured, and tested for CYP induction. Induction levels of CYP1A2, CYP2B6, and CYP3A4 mRNA were analyzed by collecting and processing PHH lysates at one site. A review of laboratory work records revealed differences in PHH seeding density and CYP inducer treatment times, which were suggested as potential sources of variability in RNA yield and CYP induction patterns. Follow-up tests confirmed that both seeding density and induction duration significantly influenced CYP mRNA expression levels, beyond lot-to-lot differences in PHH. Despite adhering to shared SOPs, these inconsistencies contributed to variability in study results. To address these challenges, we discuss key considerations for SOP development and implementation to improve reproducibility in CYP induction assays.
Authors
- Nobuhiko Kojima (ORCID: https://orcid.org/0000-0003-2555-0570)
- Chihiro Mori
- Hiroyuki Mizuguchi (ORCID: https://orcid.org/0000-0003-0594-8178)
- Daiju Yamazaki (ORCID: https://orcid.org/0000-0002-6078-5298)
- Takahiro Iwao (ORCID: https://orcid.org/0000-0002-2452-9043)
- Tamihide Matsunaga (ORCID: https://orcid.org/0000-0002-2083-1974)
- Masaki Nishikawa
- Yukiko Ueyama-Toba
- Ikue Mihara
- Kazuo Takayama (ORCID: https://orcid.org/0000-0002-1132-2457)
- Hiroko Toyoda (ORCID: https://orcid.org/0000-0002-0188-6574)
- Kosuke Inamura
- Yuzuru Ito (ORCID: https://orcid.org/0000-0001-7923-865X)
- Yu‐suke Torisawa (ORCID: https://orcid.org/0000-0002-6808-7654)
- Yuki Nishida (ORCID: https://orcid.org/0000-0001-5827-7406)
- Takafumi Akabane
- Yasuhiko Aiki
- Seiichi Ishida
- Yoko Sakai
- Yasuyuki Sakai (ORCID: https://orcid.org/0000-0001-7734-5398)
- Y. Yakabe
- Shinichiro Horiuchi
- Ayaka Nozue
Institutions
- University of Tsukuba (JP)
- Kyoto University (JP)
- Nagoya City University (JP)
- Photonics Electronics Technology Research Association (JP)
- National Institute of Health Sciences (JP)
- National Institute of Advanced Industrial Science and Technology (JP)
- The University of Tokyo (JP)
- Yokohama City University (JP)
- Osaka University of Pharmaceutical Sciences (JP)
- The University of Osaka (JP)
Publication Details
- Journal
- European Journal of Cell Biology
- Published
- 2025-06-09
- DOI
- https://doi.org/10.1016/j.ejcb.2025.151497
- Citations
- 1
- Primary Topic
- Pharmacogenetics and Drug Metabolism
- Type
- article
- Field-Weighted Citation Impact
- 1.56
Funders
- Japan Agency for Medical Research and Development