Single-step isolation of extracellular vesicles by size-exclusion chromatography
Single-step isolation of extracellular vesicles by size-exclusion chromatography
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DOI：
10.3402/jev.v3.23430
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发表时间：
2014-01-01
影响因子：
16
通讯作者：
Nieuwland, Rienk
中科院分区：
文献类型：
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作者：
Boing, Anita N.;van der Pol, Edwin;Nieuwland, Rienk
背景：由于蛋白质和脂蛋白的存在，从血浆中分离细胞外囊泡是一个挑战。使用差速离心或密度梯度超速离心分离囊泡分别导致蛋白质聚集物等污染物的共同分离以及囊泡与脂蛋白的不完全分离。目的：开发从人体体液中分离囊泡的单步方案。方法：将来自血小板浓缩物的无血小板上清液加载到琼脂糖凝胶上 CL-2B 柱进行尺寸排阻色谱（SEC；n = 3）。通过纳米颗粒跟踪分析、电阻脉冲传感、流式细胞术和透射电子显微镜收集和分析级分。测量每个级分中高密度脂蛋白胆固醇 (HDL) 和蛋白质的浓度。结果：级分 9-12 含有最高浓度的大于 70 nm 的颗粒和血小板衍生囊泡（分别占所有收集级分中存在总量的 46% 96 和 61% 92），但 HDL 含量低于 5%，蛋白质含量低于 1%（分别为 4.8% 91 和 61% 92）。 分别为 0.65% 90.3）。 HDL 主要存在于级分 18-20 中（占总数的 32% 92），蛋白质主要存在于级分 19-21 中（占总数的 36% 92）。与起始材料相比，级分 9-12 中血小板源性囊泡的回收率为 43% 923，与 HDL 和蛋白质相比，富集度为 8 倍和 70 倍。结论：SEC 可以从血小板浓缩物的无血小板上清液中有效分离出直径大于 70 nm 的细胞外囊泡。 SEC 的应用将改善细胞外囊泡的尺寸、结构和功能特性的研究。
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.