LAUSR

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.