50×10⁶ MSC(M) from 3 donors were expanded on CellStart-coated plastic microcarriers with S/X-free StemPro culture medium. In the end of the EVs conditioning phase (last 2 days), at days 9 and 13, the MSC-CM from FB and FB/CP cultures, respectively, were separated from the cell-containing microcarriers, streamed through a 0.22 µm filter and stored at -80ºC for EVs isolation and characterization. Results are presented as mean ± SEM. MSC-EV: mesenchymal stromal cell-derived extracellular vesicles, MSC-CM: mesenchymal stromal cell-derived conditioned medium, PPR: particle to protein ratio, FB: fed-batch, FB combined with continuous perfusion: FB/CP, STR: stirred tank reactor

Although the total number of EVs in the FB/CP-derived MSC-CM is lower, when compared with FB-derived MSC-CM, this value is underestimated, since, due to continuous operation the EVs did not accumulate in the reactor, as happened in the cultures operated under FB mode. To assess the purity of EV samples, particle to protein ratio (PPR) was also determined by dividing the EV concentration estimated by NTA, by the total protein concentration determined with BCA protein assay. High PPR means high sample purity due to low amount of co-isolated protein contaminants. As presented in Table 1 , EV samples isolated from MSC-CM from STR cultures operated under FB and FB/CP, presented similar PPRs of (1.7±0.21) x10⁸ and (2.0±0.22) x10⁸ particle/µg protein, respectively. Table 2 summarizes all parameters of the MSC(M) expansion process and the production of its EVs in STR operated under FB or FB/CP mode.

Table 2: Comparison of different process parameters of the expansion of MSC(M) and production of MSC-EVs in STR cultures operated under FB and FB/CP modes.