Considerations for developing CYP induction assays in hepatocytes: Insights from a multilaboratory study

Abstract

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.