Heat inactivation of foetal bovine serum causes protein contamination of extracellular vesicles

Abstract

Both the release of extracellular vesicles (EVs) in cell cultures and the cargo that these EVs carry 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 on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and heat inactivation were evaluated, including FBS that was ultracentrifuged but not heat-inactivated, FBS that was heat inactivated before EV depletion, and FBS that was heat inactivated after EV depletion. The FBS samples were then added to the culture media of three melanoma cell lines, and after 72 h both large and small EVs were isolated by differential UC. We demonstrated by transmission electron microscopy, protein measurement, and quantification of the number of particles that heat inactivation performed after EV depletion reduced the purity of small EVs but had no effect on large EV purity. Quantitative mass spectrometry analysis of FBS-derived small EVs showed that the EV protein content was different when FBS was heat inactivated after EV depletion compared to EVs isolated from FBS that was not heat inactivated or that was heat inactivated prior to EV depletion. Moreover, several of the quantified proteins were wrongly attributed to be of human origin because the EVs were of obvious bovine origin. Our results demonstrated that proteins of bovine origin coming from FBS-derived EVs could mistakenly be attributed to human cell-derived EVs in EV proteomic studies. Moreover, we concluded that heat inactivation performed after EV depletion induced the release of proteins that might contaminate EV samples, and the recommendation is therefore to always perform heat inactivation prior to EV depletion.