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Figure 1. Immunophenotypic characterization of ASCs and biophysical characterization of derived EVs. (A) Flow cytometry analysis of ASCs demonstrating a typical MSC immunophenotype, with strong expression (> 95% positive cells) of canonical MSC markers CD44, CD73, and CD90, and minimal expression (< 5%) of hemato-endothelial markers CD31 and CD45. Representative cytograms are shown; (B) Representative SSC/CFSE dot plots illustrating the sorting strategy for ASC-EVs according to a previously published protocol. CFSE-positive events correspond to sorted sASC-EVs, whereas CFSE-negative events represent excluded particles; the mean recovery of sASC-EVs across five independent experiments was 10% ± 3%; (C) Size distribution of ASC-EVs and sASC-EVs determined by NTA, showing comparable mean particle sizes (ASC-EVs: 157 ± 7 nm; sASC-EVs: 161 ± 4 nm) and median diameters (D50: 144 ± 5 nm and 149 ± 8 nm, respectively); (D) NTA-derived particle concentration and size distribution profiles of ASC-EVs and sASC-EVs, demonstrating overlapping distributions and no statistically significant differences between preparations. Data are presented as mean ± SD, n = 5 independent experiments; (E) Representative transmission electron microscopy images from one donor confirming the presence of vesicular structures with typical EV morphology in both ASC-EVs and sASC-EVs preparations. ASCs: Adipose-derived stromal cells; MSC: mesenchymal stromal cell; EVs: extracellular vesicles; sASC: sorted adipose-derived stromal cell; CD: cluster of differentiation; SSC: side scatter; CFSE: carboxyfluorescein succinimidyl ester; NTA: nanoparticle tracking analysis; D50: median particle diameter; SD: standard deviation.