FIGURE 6.

Workflow of different sEV collection strategies. (A) MSCs were seeded at 2,000–4,000 cells/cm² in the media αMEM+8% PL (M) and expanded for P1 or P2. After 24–96 h media was exchanged completely for the collection of sEVs in CM. In addition to αMEM+8% PL, αMEM+8% EV depl. PL was chosen as collection media for enrichment of MSC-derived sEVs. In order to exclude an impact of batch-to-batch variation of PL, the same starting batches of PL (from the same donors) were used for the experiments shown in the upper and lower row, which just differed whether or not EV depletion has been performed. Collection time was set to 24 or 48 h for αMEM+8% PL cultures and to 48 h for αMEM+8% EV depl. PL cultures. CM was harvested and stored at −80°C until isolation of sEVs. (B) αMEM+8% EV depl. PL was manufactured by UC of PL for 3 h at 120,000 × g at 4°C, whereby the pellet containing the PL-derived EV was withdrawn. (C) sEVs isolated from CM collected with αMEM+8% PL included MSC-derived and PL-derived sEVs, whereas sEVs collected with αMEM+8% EV depl. PL solely included MSC-derived sEVs. PL-derived sEVs were isolated from the media αMEM+8% PL to confirm the hypothesis.