Fig. 3.

Characterization of patient visceral (VAT) and subcutaneous adipose tissue (SAT) extracellular vesicles (EVs). AT samples from bariatric surgeries were cultured ex vivo in EV-free culture medium for several days, after which EVs were isolated by differential steps of ultracentrifugation. To evaluate how EV secretion changes over time in AT cultures, culture supernatant was collected from VAT cultures and replenished daily until cultures had been maintained for 3 days in total (samples 1d, 2d, 3d). Replenished culture medium was then incubated for 3 more days, and then collected (6d). Results include VAT ex vivo cultures of 6 patients, presented as mean + SEM (a). *p = 0.006 (Kruskal–Wallis ANOVA). All EV counts have been normalized to 1 g of VAT obtained for culturing. Sample purity and the morphology of VAT EV isolates obtained after 2 and 6 days of culture initiation were analyzed by scanning (SEM) and transmission electron microscopy (TEM) (b). Scale bars 1 µm. The possible presence of blood- and cell-derived material was studied by apolipoprotein A1 (ApoA1) and calnexin Western Blotting, respectively, from 1 and 2d AT EV samples (c). Final VAT and SAT EV samples (pooled 2d, 3d, and 6d samples) were further analyzed by CD63, β-actin, and tumor susceptibility 101 (TSG101) Western Blotting (d). The presence of common EV markers (CD63 and CD9) and AT-specific EV marker fatty acid binding protein 4 (FABP4) was further confirmed by fluorescent labelling and confocal microscopy (e)