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.

Extracellular vesicles released in response to respiratory exposures: implications for chronic disease

“…Benedikter et al (2017) found that EV from CSEstimulated pulmonary epithelial cells did not significantly modify IL-8 secretion by naïve cells. The same CSE-EV were less potent inducers of monocyte adhesion to endothelial cells than EV secreted by unexposed cells , questioning a role of CSE-induced EV in immune cell recruitment.…”

Isolation and characterization of extracellular vesicles from Caenorhabditis elegans for multi-omic analysis

“…Therefore, we filtered the supernatant through a 0.22 μm filter to remove larger particles. Recently it was reported that ultrafiltration, rather than ultracentrifugation, results in less damage to vesicles and a higher recovery rate 24 . While most ultrafiltration substrates will bind EVs, regenerated cellulose does not 25 , so we decanted and concentrated the 0.22 μm filtrate using a 10 kD mwco regenerated cellulose filter to the final volume of 1 ml at 4C (MilliporeSigma, Burlington MA, USA; Cat # UFC901008) (FIG 1B).…”

Proteomic analysis reveals procoagulant properties of cigarette smoke‐induced extracellular vesicles

“…EV isolation was performed by a combination of 10 kDa ultrafiltration and sepharose CL-4B SEC as described previously [13]. In brief, 60 ml of conditioned media were concentrated to 500 μl on an Amicon Ultra-15 Centrifugal Filter Unit with Ultracel 10 membrane (10 kDa MWCO, Merck Millipore), followed by separation of EVs and free protein by SEC on a 10 ml sepharose CL4B column into 24 fractions of 0.5 ml.…”

“…Although the airway epithelium forms the first line of contact with inhaled cigarette smoke, studies on the functions of EVs released by smoke-exposed airway epithelial cells are scarce. Previously, we have shown that airway epithelial cells secrete small EVs (mode size 110 nm) expressing the tetraspanins CD63, CD81 and CD9, at control conditions and when exposed to CSE [5,13]. In this study, we aimed to predict the functions of these EVs.…”