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

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

Research output: Contribution to journalArticleAcademicpeer-review

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.

Original languageEnglish
Article number15297
JournalScientific Reports
Volume7
Issue number1
DOIs
Publication statusPublished - 1 Dec 2017

Cite this

Benedikter, Birke J. ; Bouwman, Freek G. ; Vajen, Tanja ; Heinzmann, Alexandra C.A. ; Grauls, Gert ; Mariman, Edwin C. ; Wouters, Emiel F.M. ; Savelkoul, Paul H. ; Lopez-Iglesias, Carmen ; Koenen, Rory R. ; Rohde, Gernot G.U. ; Stassen, Frank R.M. / Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies. In: Scientific Reports. 2017 ; Vol. 7, No. 1.
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title = "Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies",
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.",
author = "Benedikter, {Birke J.} and Bouwman, {Freek G.} and Tanja Vajen and Heinzmann, {Alexandra C.A.} and Gert Grauls and Mariman, {Edwin C.} and Wouters, {Emiel F.M.} and Savelkoul, {Paul H.} and Carmen Lopez-Iglesias and Koenen, {Rory R.} and Rohde, {Gernot G.U.} and Stassen, {Frank R.M.}",
year = "2017",
month = "12",
day = "1",
doi = "10.1038/s41598-017-15717-7",
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Benedikter, BJ, Bouwman, FG, Vajen, T, Heinzmann, ACA, Grauls, G, Mariman, EC, Wouters, EFM, Savelkoul, PH, Lopez-Iglesias, C, Koenen, RR, Rohde, GGU & Stassen, FRM 2017, 'Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies' Scientific Reports, vol. 7, no. 1, 15297. https://doi.org/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. / Benedikter, Birke J.; Bouwman, Freek G.; Vajen, Tanja; Heinzmann, Alexandra C.A.; Grauls, Gert; Mariman, Edwin C.; Wouters, Emiel F.M.; Savelkoul, Paul H.; Lopez-Iglesias, Carmen; Koenen, Rory R.; Rohde, Gernot G.U.; Stassen, Frank R.M.

In: Scientific Reports, Vol. 7, No. 1, 15297, 01.12.2017.

Research output: Contribution to journalArticleAcademicpeer-review

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AU - Bouwman, Freek G.

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AU - Heinzmann, Alexandra C.A.

AU - Grauls, Gert

AU - Mariman, Edwin C.

AU - Wouters, Emiel F.M.

AU - Savelkoul, Paul H.

AU - Lopez-Iglesias, Carmen

AU - Koenen, Rory R.

AU - Rohde, Gernot G.U.

AU - Stassen, Frank R.M.

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