Movassaghi Cameron S, Meyer Jesse G
Department of Computational Biomedicine, Cedars Sinai Medical Center, Los Angeles CA 90048.
Advanced Clinical Biosystems Research Institute, Cedars Sinai Medical Center, Los Angeles CA 90048.
bioRxiv. 2025 Jun 22:2025.06.20.660816. doi: 10.1101/2025.06.20.660816.
Antibiotics are routinely added to mammalian cell culture media to prevent bacterial growth. However, the use of antibiotics in cell culture can confound downstream experimental results. While genomic and transcriptomic differences between cell cultures treated with and without antibiotics are well-documented, far fewer, if any, comprehensive proteomic comparisons on the use of antibiotics in cell culture have been performed. Here, we present a study on the proteome-wide differences of culturing HepG2 cells in antibiotic ( penicillin/streptomycin) and non-antibiotic-containing media. Using a longitudinal and crossover treatment study design, we analyzed 119 samples across nine passages and four conditions. On average, 9,374 proteins were detected per sample, and we identified 383 proteins that were differentially abundant between conditions. These changes included ribosomal and mitochondrial proteins, demonstrating that off-target effects of antibiotics on mammalian cells occur at the protein level. Linear mixed-effects modeling suggested that the proteomic impact of antibiotic treatment is strongest in the first passage after treatment and stabilizes after approximately three passages. Furthermore, initiating antibiotic treatment induced a greater number of differentially abundant proteins than discontinuing treatment. Lastly, we compared our results to existing literature on the use of common antibiotics in mammalian cell culture. We identified proteins and pathways conserved across studies, -omics layers, and cell types. We hope that this detailed proteomic survey will aid researchers in comparing cross-study or cross-condition results from antibiotic-treated mammalian cells and inform appropriate experimental designs for the use of antibiotics in cell culture.
抗生素通常被添加到哺乳动物细胞培养基中以防止细菌生长。然而，在细胞培养中使用抗生素可能会混淆下游实验结果。虽然有无抗生素处理的细胞培养物之间的基因组和转录组差异已有充分记录，但关于细胞培养中使用抗生素的全面蛋白质组学比较却少之又少（即便有也寥寥无几）。在此，我们展示了一项关于在含抗生素（青霉素/链霉素）和不含抗生素的培养基中培养HepG2细胞的全蛋白质组差异研究。采用纵向和交叉处理研究设计，我们分析了9个传代和4种条件下的119个样本。每个样本平均检测到9374种蛋白质，我们鉴定出383种在不同条件下丰度有差异的蛋白质。这些变化包括核糖体蛋白和线粒体蛋白，表明抗生素对哺乳动物细胞的脱靶效应发生在蛋白质水平。线性混合效应模型表明，抗生素处理对蛋白质组的影响在处理后的第一代最强，大约三代后趋于稳定。此外，开始抗生素处理比停止处理诱导出更多丰度有差异的蛋白质。最后，我们将我们的结果与关于在哺乳动物细胞培养中使用常见抗生素的现有文献进行了比较。我们鉴定出了在不同研究、组学层面和细胞类型中保守的蛋白质和通路。我们希望这项详细的蛋白质组学调查将有助于研究人员比较来自抗生素处理的哺乳动物细胞的跨研究或跨条件结果，并为细胞培养中抗生素的使用提供合适的实验设计参考。
