Supplementary file 1_Changes in cultivation parameters impact cytochrome P450 gene transcription in HepaRG cells: implications for in vitro toxicological assessments.docx

IntroductionThe HepaRG cell line has become a widely used model for liver toxicity testing due to the expression of cytochrome P450 enzymes essential for phase I metabolism of endogenous and exogenous compounds. As variations in expression may pose human health risks, determining CYP interactions of substances is crucial in toxicity assessments. Therefore, the use of human liver cell lines, such as HepaRG, for regulatory hazard assessment requires reproducible and stable CYP enzyme expression, despite possible influencing factors, such as seeding cell number, partial cell monolayer damage, and mRNA extraction timepoint.
MethodsTranscriptional changes of 12 major CYP genes in relation to changes in cultivation parameters were investigated. To this end, HepaRG cells were cultivated according to two different methods and analyzed by RT-qPCR. Cells were seeded at five densities per cultivation method and mRNA was extracted at two timepoints after completion of differentiation, also comparing extracts from undamaged and intentionally damaged cell monolayers.
ResultsA Bayesian regression model showed timepoint and cell number to have the most impact on transcription. Transcription was decreased at very high and very low cell numbers over recommended numbers, but this effect was strongly modulated by extraction timepoint, with transcription increasing after two additional weeks in culture. Intentional damage to the cell monolayer had marginal effects on transcription, and no evidence of an effect of cultivation method was found.
ConclusionIn summary, extraction timepoint and seeding cell number are the two critical parameters to consider before initiating a CYP expression experiment with HepaRG cells.

引言
HepaRG细胞系（HepaRG cell line）因可表达介导内源性与外源性化合物I相代谢的关键酶类——细胞色素P450酶（cytochrome P450 enzymes），已成为肝脏毒性测试的通用模型。由于基因表达的差异可能对人体健康构成风险，明确物质与CYP酶的相互作用在毒性评价中至关重要。因此，在监管机构要求的肝脏危害评估中，使用HepaRG等人类肝细胞系时，需保证CYP酶表达具备可重复性与稳定性，尽管存在诸多潜在影响因素，如接种细胞数量、细胞单层局部损伤以及mRNA提取时间点。

材料与方法
本研究针对培养参数变化对12种主要CYP基因转录水平的影响展开探究。实验采用两种不同培养方法培养HepaRG细胞，并通过逆转录实时定量PCR（RT-qPCR）进行分析。每种培养方法下，细胞以5种不同密度接种，并在分化完成后的两个时间点提取mRNA；同时对比了未受损与人为损伤的细胞单层的提取样本。

结果
贝叶斯回归模型（Bayesian regression model）分析显示，提取时间点与接种细胞数量对转录水平影响最为显著。当细胞数量超出推荐范围的极高或极低值时，转录水平会出现下降，但该效应会受到提取时间点的强烈调控：在培养额外两周后，转录水平会有所提升。人为造成的细胞单层损伤对转录水平仅存在微弱影响，未观察到培养方法对转录水平的显著作用。

结论
综上，在开展基于HepaRG细胞的CYP表达实验前，需重点关注两个关键参数：mRNA提取时间点与接种细胞数量。