Heat inactivation of fetal bovine serum increases protein contamination of extracellular vesicles

Introduction: Extracellular vesicles (EVs) released in cell cultures are influenced by the cell culture conditions, such as the use of fetal bovine serum (FBS). FBS contains EVs and it is usually depleted of EVs by ultracentrifugation (UC) and/or heat inactivated (HI). Several studies have evaluated the effect of different UC protocols for FBS by evaluating both cells and EVs. However, less is known about the effect of HI on the cells and the released EVs. The aim of this study was therefore to evaluate the effect of HI on EV purity. Methods: To determine the effect of heat inactivation, three different protocols were applied based on different combinations of: 1) UC at 118,000 × g for 18h and 2)HI at 56◦C for 30 min. The three conditions tested were: FBS ultracentrifuged but not heat inactivated (no-HI), FBS heat inactivated before UC (HI-before EV-dep), and FBS heat inactivated after EV depletion (HI-after EV-dep). The FBS was add to themedia of threemelanoma cell lines (MML1, UM22Ctr and UM22BAP1) at a final concentration of 10%. After 72h, large and small EVs were isolated by differential UC. The EV purity was determined by protein quantity, electron microscopy (EM) and nanoparticle tracking analysis (NTA). Results: The protein quantity (μg/μl) of large EVs was similar in the three conditions analyzed. On the contrary for small EVs, the protein amount was higher when the HI was performed after EV depletion as compared to HI before the UC and UC alone. However, significantly more particles were not detected in the HI-after EV-dep which resulted in a lower purity of small EVs in HI-after EV-dep illustrated by calculating the ratio of number of particles/μg proteins. Presence of contaminants (indicated by strong background) was observed in EM pictures of small EVs isolated in HI-after EV-dep condition differently from large EV samples. Summary/Conclusion: The HI of FBS induces release of contaminating elements that end up in small EVpellets if not previously removed.