(A) EVs were isolated from MSCs at P2, P3, P4, and P5 were seeded at 1E2, 5E2, 1E3, 1E4 cells/cm2. For MSCs at all passages, increase in EV production is observed per cell with decreasing seeding densities. Statistical significance was calculated using two-way ANOVA with Tukey’s multiple comparison test (* p<0.05, ** p<0.01). Vascular bioactivity of EVs from different densities (B) and different passages (C) was analyzed using a gap closure assay with endothelial cells. (B) No significant difference in gap closure was observed for EVs from MSCs seeded at 1E2 and 1E4 cells/cm2. (C) Bioactivity of EVs significantly diminishes with increasing passage. Data are representative of three independent experiments with three replicates each (n = 3). Statistical significance was calculated using two-way ANOVA with Tukey’s multiple comparison test (* p<0.05, ** p<0.01, **** p<0.0001). Figure reprinted from Patel et al., 2017 via open access.