Single-step isolation of extracellular vesicles by size-exclusion chromatography - PubMed (original) (raw)

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

Anita N Böing et al. J Extracell Vesicles. 2014.

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.

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.

Keywords: extracellular vesicles; isolation; lipoproteins; plasma; protein; size-exclusion chromatography.

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Figures

Fig. 1

Fig. 1

Image of size-exclusion chromatography column. A 10 mL syringe stacked with sepharose CL-2B for isolation of vesicles from platelet-free supernatant of platelet concentrates.

Fig. 2

Fig. 2

Presence of vesicles, protein and lipoproteins per fraction. The concentration of vesicles, protein and lipoproteins was measured in each fraction. Each bar shows the number present in a fraction as % of the total number that passed the column. The height of the bar represents the mean, the error bars the standard deviation from 3 experiments. a) Particles (larger than 70 nm) measured by NTA. b) Particles (100–400 nm) measured by RPS. c) CD61+ vesicles measured by flow cytometry. d) Lactadherin+vesicles measured by flow cytometry. e) HDL (Cholesterol) concentration measured by a colorimetric assay. f) HDL (APO A1) concentration measured by a turbidimetric assay. g) Protein concentration measured by a Bradford protein assay h) Overview of all measured results.

Fig. 3

Fig. 3

Presence of proteins, CD63 and CD9 in collected fractions. a) The presence of proteins in each fraction determined by loading 20 µL on PAGE gels. The molecular weight of albumin is 66 kDa. b,c) Presence of tetraspanins in the different fractions was studied by Western blot, with 4 µg protein used per fraction. First, the presence of CD63 was shown (53 kDa, panel b), and next the presence of CD9 was shown (24 kDa, panel c). The tetraspanin bands are indicated by arrows in panels b and c. Platelet lysate was used as positive control.

Fig. 4

Fig. 4

TEM images of fractions. Starting material and fractions, undiluted or 50-fold diluted when indicated, were analysed by TEM for the presence of particles. All images shown are representative images for the starting material (a, j) and fractions 5, 9, 10, 11, 17, 18, 19 and 20 (b–i, k–n). Scale bar is 200 nm (a–g, j–n), 500 nm (h), or 1 µm (i).

Fig. 5

Fig. 5

Recovery and enrichment. The recovery and enrichment relative to the starting material, in the vesicle-containing fractions (9, 9–10, 9–11, 9–12) are shown. a) Recovery of vesicles, protein and HDL (cholesterol) in the vesicle-containing fractions. b) Enrichment factor of vesicle to protein. c) Enrichment factor of vesicle to HDL (cholesterol).

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