Importance of exosome depletion protocols to eliminate functional and RNA‐containing extracellular vesicles from fetal bovine serum

TLDR

Extracellular vesicles, including exosomes, can transfer functional molecules between cells, and fetal bovine serum used in cell culture contains significant quantities of such vesicles. The study aimed to assess whether FBS‑derived EVs alter cultured cell phenotype and to evaluate the effectiveness of different depletion protocols. FBS EVs induced a migratory phenotype in A549 cells, carried protected RNA, and were largely removed (≈95 %) by an 18‑hour centrifugation, whereas a 1.5‑hour protocol was inadequate, demonstrating that FBS EVs influence cell behavior but can be virtually eliminated with prolonged ultracentrifugation.

Abstract

Extracellular vesicles (EVs), including the nano-sized exosomes, have the capacity to transfer multiple functional molecules between cells. In cell culture experiments, fetal bovine serum (FBS) is often used to supplement cell culture medium as a nutrient, but it is important to know that the FBS also contain significant quantities of EVs. The aim of the current study was to determine whether the FBS EVs can influence cultured cell phenotype, and secondly to determine the efficiency of FBS-EV elimination protocols. Firstly, FBS that had not been depleted of EVs induced a migratory phenotype in a lung cancer epithelial cell line (A549 cells), an effect that could be mimicked by isolated FBS EVs alone. FBS-derived EVs also contained RNA, which was protected from consecutive proteinase K and RNase A treatment. Comparison of common isolation protocols suggested that an 18-hour centrifugation period eliminates approximately 95% of RNA-containing FBS EVs, whereas a 1.5-hour protocol is insufficient. In conclusion, this study shows that FBS EVs substantially influence cultured cell behaviour, but also that they can be virtually removed by an 18-hour ultracentrifugation protocol.