title: Single-step isolation of extracellular vesicles by size-exclusion chromatography.
abstract: 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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Department of Clinical Chemistry, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Anita N
authors.initials: AN
authors.lastname: Böing
authors.affiliation: Department of Clinical Chemistry, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands ; Department of Biomedical Engineering and Physics, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Edwin
authors.initials: E
authors.lastname: van der Pol
authors.affiliation: Department of Clinical Chemistry, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Anita E
authors.initials: AE
authors.lastname: Grootemaat
authors.affiliation: Department of Clinical Chemistry, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands ; Department of Biomedical Engineering and Physics, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Frank A W
authors.initials: FA
authors.lastname: Coumans
authors.affiliation: Department of Clinical Chemistry, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Auguste
authors.initials: A
authors.lastname: Sturk
authors.affiliation: Department of Clinical Chemistry, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Rienk
authors.initials: R
authors.lastname: Nieuwland
keywords: extracellular vesicles; isolation; lipoproteins; plasma; protein; size-exclusion chromatography