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. Overall design: 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.