Morphological, size, and protein characterization of stored BDMSC EVs. (A) Representative concentration and size distribution profile of isolated BDMSC EVs as assessed by NTA. (B) Mode size (diameter) changes under varying storage conditions of BDMSC EVs at day 0, 7 and 28. (C) Mode size (diameter) changes of BDMSC EVs after 1 freeze-thaw (FT1) and 5 freeze-thaw cycles (FT5) compared to day 0. (D) TEM images of isolated BDMSC EVs immediately after isolation or after lyophilization and reconstitution. (E) Immunoblot analysis of BDMSC EV markers TSG101 and CD63 after storage at room temperature (RT), 4°C, −20°C, −80°C, or freeze-thaw cycles FT1 and FT5. (F) Protein concentrations of BDMSC EVs under varying storage conditions of RT, 4°C, −20°C, or −80°C after 7 or 28 days as compared to day 0. Concentration was determined via BCA assay. (G) Protein concentrations of BDMSC EVs after FT1 or FT5 compared to day 0, assessed via BCA assay. (H) Changes in protein concentration of BDMSC EVs following lyophilization and storage at RT for 7 or 28 days compared to pre-lyophilization. Data are representative of three independent experiments (n=3); All values are expressed as mean ± standard deviation (*p < 0.05, **p < 0.01).