Autologous MSCs were collected from abdominal fat (5-10g) of 4 female domestic pigs. Adipose tissue was digested in collagenase-H for 45min, filtered, and cultured for 3 weeks in advanced MEM medium (Gibco/Invitrogen) supplemented with 5% platelet lysate (PLTmax, Mill Creek Life Sciences, Rochester, MN) in 37 degree/5% CO2. The 3rd passage was collected and kept in Gibco Cell Culture Freezing Medium (Life Technologies) at -80°C for in-vitro phenotype/function analysis. We avoided the use of any animal products (beyond porcine MSCs) in our cell culture procedures to approximate a clinical-grade tissue culture product. Cellular phenotype was examined in-vitro with immuno-fluorescent staining of porcine MSCs positive for CD90, CD44, and CD105, as previously described5 (link), 15 (link), 16 (link) and consistent with our experience with human MSCs17 , 18 .
EVs were isolated from supernatants of 10×106 MSCs and cultured for 48h in advanced MEM medium without supplements. After centrifugation at 2000g, cell-free supernatants were ultra-centrifuged at 100,000g for 1h at 4°C, washed in serum-free medium containing HEPES 25mM and submitted to a second ultracentrifugation. EVs were collected and characterized based on the expression of both microvesicle (ß1-integrins, CD73, and CD40) and exosome (CD9 and CD81) markers using fluorescence activated cell sorting (FACS)19 (link).
EVs were isolated from supernatants of 10×106 MSCs and cultured for 48h in advanced MEM medium without supplements. After centrifugation at 2000g, cell-free supernatants were ultra-centrifuged at 100,000g for 1h at 4°C, washed in serum-free medium containing HEPES 25mM and submitted to a second ultracentrifugation. EVs were collected and characterized based on the expression of both microvesicle (ß1-integrins, CD73, and CD40) and exosome (CD9 and CD81) markers using fluorescence activated cell sorting (FACS)19 (link).