Single-step isolation of extracellular vesicles by size-exclusion chromatography

Background: Isolation of extracellular vesicles from plasma is a challenge due to the presence of proteins and lipoproteins. Isolation of vesicles using differential centrifugation or density-gradient ultracentrifugation results in co-isolation of contaminants such as protein aggregates and incomplete separation of vesicles from lipoproteins, respectively. Aim: To develop a single-step protocol to isolate vesicles from human body fluids. Methods: Platelet-free supernatant, derived from platelet concentrates, was loaded on a sepharose CL-2B column to perform size-exclusion chromatography (SEC; n = 3). Fractions were collected and analysed by nanoparticle tracking analysis, resistive pulse sensing, flow cytometry and transmission electron microscopy. The concentrations of high-density lipoprotein cholesterol (HDL) and protein were measured in each fraction. Results: Fractions 9-12 contained the highest concentrations of particles larger than 70 nm and plateletderived vesicles (46% 96 and 61% 92 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8% 91 and 0.65% 90.3, respectively). HDL was present mainly in fractions 18-20 (32% 92 of total), and protein in fractions 19-21 (36% 92 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43% 923 in fractions 9-12, with an 8- fold and 70-fold enrichment compared to HDL and protein. Conclusions: SEC efficiently isolates extracellular vesicles with a diameter larger than 70 nm from platelet-free supernatant of platelet concentrates. Application SEC will improve studies on the dimensional, structural and functional properties of extracellular vesicles.