Sahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational Medicine, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
Department of Biomedicine, Neurosciences and Advanced Diagnostics (Bi.N.D), University of Palermo, Palermo, Italy.
J Extracell Vesicles. 2024 Jan;13(1):e12408. doi: 10.1002/jev2.12408.
The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion.
细胞培养中细胞外囊泡 (EVs) 的释放及其分子货物可以受到细胞培养条件的影响，例如胎牛血清 (FBS) 的存在。虽然有几项研究评估了通过超速离心 (UC) 去除 FBS 衍生 EV 的效果，但对于 FBS 热失活 (HI) 对细胞衍生 EV 的影响知之甚少。为了评估这一点，评估了三种基于 UC 和 HI 去除 EV 的不同组合的方案，包括未 HI 的 FBS（no-HI FBS）、HI 前 EV 耗尽的 FBS（HI-before EV-depl FBS）和 HI 后 EV 耗尽的 FBS（HI-after EV-depl FBS）。我们从以三种不同方式处理的 FBS 中分离出大 (L-EVs) 和小 EVs (S-EVs)，发现 HI-after EV-depl FBS 的 S-EV 沉淀比 no-HI FBS 和 HI-before EV-depl FBS 的 S-EV 沉淀更大。透射电子显微镜、蛋白质定量和颗粒数评估表明，HI-after EV-depl 显著增加了 S-EV 的蛋白质含量，但对 L-EV 没有显著影响。因此，用 HI-after EV-depl FBS 补充的培养基培养的三种细胞系中分离的 S-EVs 的蛋白质数量显著增加。定量质谱分析 FBS 衍生的 S-EVs 表明，与从 no-HI FBS 和 HI-before EV-depl FBS 中分离的 EV 相比，EV 耗尽后 HI 的 FBS 中的 EV 蛋白含量不同。此外，我们表明，一些定量蛋白质可以归因于人类起源，从而证明 FBS 牛蛋白可能会错误地归因于人类细胞衍生的 EV。我们得出结论，EV 耗尽后 FBS 的 HI 会导致蛋白质组发生变化，与 EV 共分离的分子在用于后续细胞培养时会污染 EV。因此，我们建议始终在 EV 耗尽前对 FBS 进行 HI。
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