Mesenchymal stem cells were isolated from swine subcutaneous abdominal fat tissue, which was digested in collagenase-H, filtered, and cultured for 3 weeks in advanced MEM medium (Gibco/Invitrogen) supplemented with 5% platelet lysate, as previously described [10 (link)]. At passage 3, MSCs were collected and cellular phenotype confirmed by the expression of the MSCs markers CD73, CD105, CD44, and CD90, as well as by their capacity for tri-lineage differentiation, as previously shown [16 (link), 17 (link)].
Extracellular vesicles were isolated from passage 3-MSC supernatants (10 × 106 cells) by ultracentrifugation, as previously described [8 (link)–10 (link), 18 (link), 19 (link)]. Samples were centrifuged (2000g for 20 min), and the supernatant collected and subsequently centrifuged (100,000g for 1 h) at 4 °C. EVs were collected, suspended in wash buffer medium 199, centrifuged once more (100,000g for 1 h), and characterized based on the expression of common EV (CD9 and CD63) and MSC surface markers, as previously shown [8 (link), 10 (link), 20 (link)].
Extracellular vesicles were isolated from passage 3-MSC supernatants (10 × 106 cells) by ultracentrifugation, as previously described [8 (link)–10 (link), 18 (link), 19 (link)]. Samples were centrifuged (2000g for 20 min), and the supernatant collected and subsequently centrifuged (100,000g for 1 h) at 4 °C. EVs were collected, suspended in wash buffer medium 199, centrifuged once more (100,000g for 1 h), and characterized based on the expression of common EV (CD9 and CD63) and MSC surface markers, as previously shown [8 (link), 10 (link), 20 (link)].
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