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.

中文翻译：

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EV耗尽后进行的胎牛血清热灭活影响细胞源性细胞外囊泡的蛋白质组

细胞培养物中细胞外囊泡 (EV) 及其分子货物的释放可能受到细胞培养条件（例如胎牛血清 (FBS) 的存在）的影响。尽管一些研究评估了通过超速离心 (UC) 去除 FBS 衍生的 EV 的效果，但关于 FBS 热灭活 (HI) 对细胞衍生的 EV 的影响知之甚少。为了评估这一点，评估了基于 UC 和 HI 消除 EV 的不同组合的三种方案，包括 FBS 超速离心但不热灭活 (no-HI FBS)、EV 消除前 FBS 热灭活 (HI-before EV-depl FBS)、 EV 耗尽后 FBS 热失活（HI-EV-depl FBS 后）。我们从以三种不同方式处理的 FBS 中分离出大型 EV（L-EV）和小型 EV（S-EV），我们发现从 HI-EV-depl FBS 中得到的 S-EV 颗粒比 S-EV 颗粒更大来自 no-HI FBS 和 HI-before EV-depl FBS。透射电子显微镜、蛋白质定量和颗粒数评估表明，EV-depl 后的 HI 显着增加了 S-EV 的蛋白质含量，但对 L-EV 没有显着影响。因此，从在补充有 HI-EV-depl FBS 的培养基中培养的三种细胞系中分离的 S-EV 的蛋白质量显着增加。FBS 衍生的 S-EV 的定量质谱分析表明，与从无 HI FBS 和 EV-depl FBS 之前的 HI 分离的 EV 相比，EV 耗尽后 FBS 为 HI 时，EV 蛋白含量不同。此外，我们发现一些定量的蛋白质可以归因于人类来源，从而证明FBS牛蛋白质可以错误地归因于人类细胞衍生的EV。我们得出的结论是，EV 耗尽后进行的 FBS HI 会导致蛋白质组发生变化，其中的分子与 EV 共分离，并且在随后的细胞培养中使用时可能会污染 EV。因此，我们的建议是始终在 EV 耗尽之前执行 FBS HI。