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. 2021 Jan 24;22(3):1142. doi: 10.3390/ijms22031142

Figure 2.

Figure 2

Formation of artificial MVs. Three separate approaches for the artificial formation of MVs. (A) The 3D mesenchymal stem cell (MSC)-culturing technique uses polyethylene glycol hydrogels to form spheroid MSCs. MSC-spheroids are subsequently cultured for 7 days on a 30-rpm orbital-shaker (3D w/shaking). Scalable pro-duction of MSC-derived MVs can be achieved by this method. (B) The ultracentrifugation approach. This includes numerous centrifugation steps. MSCs are first cultured with complete media supplemented with 15% vesicle-depleted fetal bovine serum (FBS) for 3 days. MSCs are then centrifuged at 120,000× g for 18 h to deplete extra-cellular vesicles from FBS. The cell solution is centrifuged at 300× g for 30 min at 4 °C to remove whole cells and large debris. Supernatants are then centrifuged at 16,500× g for 20 min at 4 °C to collect the MV fraction [149]. (C) Cytochalasin B-induced MVs. This approach depends on the incubation of MSCs with cytochalasin-B to obtain MVs.