title: Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum.
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.
authors.affiliation: Krefting Research Centre, Institute of Medicine at the Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Ganesh Vilas
authors.initials: GV
authors.lastname: Shelke
authors.affiliation: Krefting Research Centre, Institute of Medicine at the Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Lässer
authors.affiliation: Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk 790-784, Republic of Korea.
authors.forename: Yong Song
authors.initials: YS
authors.lastname: Gho
authors.affiliation: Krefting Research Centre, Institute of Medicine at the Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
authors.forename: Jan
authors.initials: J
authors.lastname: Lötvall
keywords: Depletion protocol; Exosomes; Extracellular vesicles; Fetal bovine serum; RNA