title: Size-Exclusion Chromatography-based isolation minimally alters Extracellular Vesicles' characteristics compared to precipitating agents.
abstract: Extracellular vesicles (EVs) have become an attractive field among the scientific community. Yet, a major challenge is to define a consensus method for EVs isolation. Ultracentrifugation has been the most widely used methodology but rapid methods, including Size Exclusion Chromatography (SEC) and/or precipitating agents such as Polyethylene glycol (PEG) or PRotein Organic Solvent PRecipitation (PROSPR) have emerged. To evaluate the impact of these different methods on the resulting EV preparations, plasma EVs were isolated using SEC, PEG and PROSPR, and their total protein content, NTA and Cryo-electron microscopy profiles, and EV-markers were compared. Also, their effect on recipient cells was tested. Low protein content and Cryo-EM analysis showed that SEC removed most of the overabundant soluble plasma proteins, which were not removed using PEG and partially by PROSPR. Moreover, only SEC allowed the detection of the EV-markers CD9, CD63 and CD81, LGALS3BP and CD5L, suggesting a putative interference of the precipitating agents in the structure/composition of the EVs. Furthermore, PEG and PROSPR-based EV isolation resulted in reduced cell viability in vitro. These results stress that appropriate EV-isolation method should be considered depending on the forthcoming application of the purified EVs.
authors.affiliation: REMAR-IVECAT Group, Health Science Research Institute Germans Trias i Pujol, Can Ruti Campus, Badalona, Spain.|Department of Pathology, Hospital Universitari and Health Sciences Research Institute Germans Trias i Pujol, Universitat Autònoma de Barcelona, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Gámez-Valero
authors.affiliation: REMAR-IVECAT Group, Health Science Research Institute Germans Trias i Pujol, Can Ruti Campus, Badalona, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Monguió-Tortajada
authors.affiliation: REMAR-IVECAT Group, Health Science Research Institute Germans Trias i Pujol, Can Ruti Campus, Badalona, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Carreras-Planella
authors.affiliation: REMAR-IVECAT Group, Health Science Research Institute Germans Trias i Pujol, Can Ruti Campus, Badalona, Spain.
authors.forename: Marcel la
authors.initials: Ml
authors.lastname: Franquesa
authors.affiliation: Department of Pathology, Hospital Universitari and Health Sciences Research Institute Germans Trias i Pujol, Universitat Autònoma de Barcelona, Spain.
authors.forename: Katrin
authors.initials: K
authors.lastname: Beyer
authors.affiliation: REMAR-IVECAT Group, Health Science Research Institute Germans Trias i Pujol, Can Ruti Campus, Badalona, Spain.|Nephrology Service, Germans Trias i Pujol University Hospital, Badalona, Spain.
authors.forename: Francesc E
authors.initials: FE
authors.lastname: Borràs
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival; D011232:Chemical Precipitation; D002850:Chromatography, Gel / Q000379:methods*; D020285:Cryoelectron Microscopy; D000067128:Extracellular Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D005434:Flow Cytometry; D006801:Humans; D011506:Proteins / Q000378:metabolism
chemical_list: D011506:Proteins