Extracellular vesicles (EVs) are important intercellular communication messengers. Half of the published studies in the field are in vitro cell culture based in which bovine serum in various concentrations and forms is used to facilitate the production of extracellular vesicles. ‘Exosome depleted serum’ is the type of bovine serum most widely used in the production of human EVs. Herein, we demonstrate that, despite the initial caution raised in 2014 about the persistence of bovine EVs, ‘exosome depleted serum’ was still used in 46% of publications on human or rodent EVs between 2015 and 2019. Using nanoparticle tracking analysis combined with detergent lysis of vesicles as well as bovine CD9 ELISA, we show that there were approximately 5.33 x 10⁷/mL of bovine EVs remaining in the ‘exosome depleted serum’. Importantly, the ‘exosome depleted serum’ was relatively enriched in small EVs by approximately 2.7-fold relative to the large EVs compared to that in the original serum. Specifically, the percentage of small EVs in total vesicles had increased from the original 48% in the serum before ultracentrifugation to 92% in the ‘exosome depleted serum’. Furthermore, the pervasive bovine EVs carried over by the ‘exosome depleted serum’, even when the lowest concentration (0.5%) was used in cell culture, resulted in a significant contamination of human EVs in cell culture conditioned medium. Our findings indicate that the use ‘exosome depleted serum’ in cell culture-based studies may introduce artefacts into research examining the function of human and rodent EVs, in particular those involving EV miRNA. Thus, we appeal to the researchers in the EV field to seriously reconsider the practice of using ‘exosome depleted serum’ in the production of human and other mammalian EVs in vitro.

细胞外囊泡 (EV) 是重要的细胞间通讯信使。该领域已发表的研究中有一半是基于体外细胞培养的，其中使用各种浓度和形式的牛血清来促进细胞外囊泡的产生。“外泌体耗尽血清”是最广泛用于人类 EV 生产的牛血清类型。在此，我们证明，尽管 2014 年最初对牛 EV 的持久性提出了警告，但在 2015 年至 2019 年间，46% 的人类或啮齿动物 EV 出版物中仍使用“外泌体耗尽血清”。 使用纳米颗粒跟踪分析结合洗涤剂囊泡裂解以及牛 CD9 ELISA，我们显示大约有 5.33 x 10 ⁷/mL 牛 EV 残留在“外泌体耗尽血清”中。重要的是，与原始血清相比，“外泌体耗尽血清”在小 EV 中相对富集约 2.7 倍于大 EV。具体来说，总囊泡中小 EV 的百分比从超速离心前血清中的原始 48% 增加到“外泌体耗尽血清”中的 92%。此外，即使在细胞培养中使用最低浓度 (0.5%) 时，“外泌体耗尽血清”携带的普遍牛 EV 也会导致细胞培养条件培养基中的人 EV 受到严重污染。我们的研究结果表明，在基于细胞培养的研究中使用“外泌体耗尽血清”可能会将人工制品引入检查人类和啮齿动物 EV 功能的研究中，特别是那些涉及 EV miRNA 的。因此，我们呼吁 EV 领域的研究人员认真重新考虑在人类和其他哺乳动物 EV 生产中使用“外泌体耗尽血清”的做法体外。