MSCs were collected from swine subcutaneous abdominal fat tissue (5–10g), digested in collagenase-H, filtered, and cultured for 3 weeks in advanced MEM medium (Gibco/Invitrogen) supplemented with 5% platelet lysate. The third passage was collected and kept in Gibco Cell Culture Freezing Medium. Cellular phenotype was examined by the expression of the MSCs markers CD73 and CD105.
EVs were isolated from MSC supernatants (10×106 cells) by the ultracentrifugation method, as previously described [4 (link)]. Samples were centrifuged (2,000g for 20min), and the supernatant collected and subsequently centrifuged (100,000g for 1h) at 4°C. EVs were collected, suspended in wash buffer medium 199, and centrifuged one more time (100,00g for 1hr).
MSCs and their daughters EVs were examined on transmission electron microscopy (JEOL 1200 EXII, Mayo Clinic’s electron microscopy core). In addition, negative staining of MSC supernatant with 2% uranyl acetate was performed to examine EV morphology.
EVs were then characterized based on the expression of both EV (CD9 and CD29) and MSC (CD73 and CD105) surface markers by western blot (AbD Serotec cat#: MCA1189 and AA120-175, and Abcam cat#: ab115289 and ab135528).
EVs were isolated from MSC supernatants (10×106 cells) by the ultracentrifugation method, as previously described [4 (link)]. Samples were centrifuged (2,000g for 20min), and the supernatant collected and subsequently centrifuged (100,000g for 1h) at 4°C. EVs were collected, suspended in wash buffer medium 199, and centrifuged one more time (100,00g for 1hr).
MSCs and their daughters EVs were examined on transmission electron microscopy (JEOL 1200 EXII, Mayo Clinic’s electron microscopy core). In addition, negative staining of MSC supernatant with 2% uranyl acetate was performed to examine EV morphology.
EVs were then characterized based on the expression of both EV (CD9 and CD29) and MSC (CD73 and CD105) surface markers by western blot (AbD Serotec cat#: MCA1189 and AA120-175, and Abcam cat#: ab115289 and ab135528).
