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.