Process Parameter STR Feeding Operation Mode STR Feeding Operation Mode
FB FB/CP
MSC(M) Inicial cell adhesion (24h) (%) 34±5.8 40±5.9
Maximal cell concentration (x105 Cells/mL) 2.0±0.51 4.1±0.90
Maximal cell number (x108 Cells) 3.0±0.12 (day 8) 5.3±0.32 (day 12)
FI in total cell number 16±2.1 24±5.5
Specific growth rate (day-1) 0.6±0.1 0.5±0.1
Duplication time (day) 1.3±0.21 1.5±0.20
MSC-EV EV Mean size (nm) 163±5.27 162±4.44
EV Mode size (nm) 134±4.23 137±6.92
EV concentration (x1011 EVs/ml) 2.4±0.35 3.0±0.48
Total EV (x1014 EVs) 4.2±0.61 3.9±0.62
PPR (x108 particle/µg protein) 1.7±0.21 2.0±0.22
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, FI: Fold increase in total cell number. 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, FI: Fold increase in total cell number. 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, FI: Fold increase in total cell number. 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, FI: Fold increase in total cell number.