Cell passage number: an intrinsic factor influencing the reproducibility of cell experiments

The reproducibility of scientific research has been increasingly challenged in recent years. While extrinsic factors (e.g., cross-contamination, mycoplasma infection, serum variability) are well-studied, the role of intrinsic attributes like cell passage number remains underexplored. Using two tumor cell lines (ACHN and Renca), we employed RNA sequencing to analyze transcriptomic dynamics across passages (P3 to P39). Results revealed a nonlinear transcriptomic shift: mid-passage cells (P10/P11, P17) showed heightened activity in cell cycle, metabolism, and stress response, whereas low (P3) and high passages (P24/P39) exhibited stable and similar profiles. KEGG analysis indicated significant alterations in signaling, immune, and metabolic pathways during passaging. This study demonstrates that passage number shapes transcriptional landscapes and impacts experimental reproducibility. We advocate for strict passage control and explicit reporting of passage information to enhance reliability. These findings underscore the need for standardized cell culture practices to improve research reproducibility. In this study, we focus on cell passage number to investigate its potential impact on the reproducibility of cell-based experimental results. We selected two commonly used tumor cell lines, Renca and ACHN, as research models. After thawing from cryopreservation (designated as P0), cells were continuously passaged at a 1:4 ratio up to P39. During this process, we collected cells at passages P3, P10/11, P17, P24, and P39 for RNA sequencing analysis.

近年来，科学研究的可重复性正面临日益严峻的挑战。尽管外源性因素（如交叉污染、支原体感染、血清异质性）已得到充分研究，但诸如细胞传代次数（cell passage number）这类内源性特征的作用仍未得到充分探索。本研究以ACHN和Renca两种肿瘤细胞系为研究模型，采用RNA测序（RNA sequencing）技术分析了从P3至P39传代过程中的转录组动态变化。结果显示转录组呈现非线性偏移：中传代细胞（P10/P11、P17）的细胞周期、代谢及应激反应通路活性显著上调，而低传代（P3）与高传代（P24/P39）细胞的转录组谱则较为稳定且相似。京都基因与基因组百科全书（KEGG）分析表明，细胞传代过程中信号通路、免疫通路及代谢通路均发生显著改变。本研究证实，细胞传代次数可塑造细胞的转录组特征，并对实验可重复性产生影响。我们倡导严格管控细胞传代流程，并明确报告传代相关信息，以提升研究可靠性。上述研究结果凸显了标准化细胞培养操作对于改善研究可重复性的必要性。本研究聚焦细胞传代次数，探究其对基于细胞的实验结果可重复性的潜在影响。我们选取了两种常用肿瘤细胞系Renca与ACHN作为研究模型。细胞从冻存复苏后（记为P0），以1:4的传代比例持续扩增至P39。在此期间，我们分别于P3、P10/11、P17、P24及P39传代时收集细胞，用于RNA测序分析。