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

Peer-reviewed article
A.N. Böing, E. van der Pol, A.E. Grootemaat, F.A.W. Coumans, A. Sturk and R. Nieuwland
Published September 8, 2014
Journal of Extracellular Vesicles
23430: 1-11
Extracellular vesicles, isolation, lipoproteins, plasma, protein, size-exclusion chromatography
Boing 2014 JEV Single-step isolation.pdf (1,782 kB)
Boing 2014 JEV Single-step isolation Supp.pdf (277 kB)


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 platelet-derived vesicles (46±6 and 61±2 of totals present in all collected fractions, respectively), but less than 5% of HDL and less than 1% of protein (4.8±1 and 0.65±0.3, respectively). HDL was present mainly in fractions 18-20 (32±2 of total), and protein in fractions 19-21 (36±2 of total). Compared to the starting material, recovery of platelet-derived vesicles was 43±23 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.