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

小泡 纳米粒子跟踪分析 差速离心 大小排阻色谱法 色谱法 超离心机 离心 化学 血小板 生物化学 微泡 生物 小RNA 基因 免疫学
作者
Anita N. Böing,Edwin van der Pol,Anita E. Grootemaat,Frank A. W. Coumans,Auguste Sturk,Rienk Nieuwland
出处
期刊:Journal of extracellular vesicles [Taylor & Francis]
卷期号:3 (1) 被引量:979
标识
DOI:10.3402/jev.v3.23430
摘要

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.
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