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Sci Rep. 2017 Nov 10;7(1):15297. doi: 10.1038/s41598-017-15717-7.

Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies.

Author information

1
Department of Medical Microbiology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, PO box 5800, 6202AZ, Maastricht, The Netherlands.
2
Department of Respiratory Medicine, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, PO box 5800, 6202AZ, Maastricht, The Netherlands.
3
Department of Human Biology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, PO box 5800, 6202AZ, Maastricht, The Netherlands.
4
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands, PO box 616, 6200 MD, Maastricht, The Netherlands.
5
Department of Medical Microbiology & Infection Control, VU University Medical Center, Van Boechorststraat 7, 1081BT, Amsterdam, The Netherlands.
6
Microscopy Core Lab, M4I Nanoscopy division, FHML, Maastricht University, Universiteitssingel 50, G0.201, 6229 ER, Maastricht, The Netherlands.
7
Medical clinic I, Department of Respiratory Medicine, Goethe University Hospital, Frankfurt/Main, Germany.
8
Department of Medical Microbiology, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center, PO box 5800, 6202AZ, Maastricht, The Netherlands. f.stassen@maastrichtuniversity.nl.

Abstract

Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules.

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