Ultrafiltration Combined with Size Exclusion Chromatography Efficiently Isolates Extracellular Vesicles from Cell Culture Media for Compositional and Functional Studies

Overview

Journal Sci Rep

Specialty Science

Date 2017 Nov 12

PMID 29127410

Citations 149

Authors

Birke J Benedikter

Freek G Bouwman

Tanja Vajen

Alexandra C A Heinzmann

Gert Grauls

Edwin C Mariman

Emiel F M Wouters

Paul H Savelkoul

Carmen Lopez-Iglesias

Rory R Koenen

Gernot G U Rohde

Frank R M Stassen

Affiliations

Soon will be listed here.

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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