Figure 2: Characterization of human MSC-derived extracellular vesicles MSC-EVs produced in a fully-controlled STR operated under FB or FB/CP operation mode. 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. (A) Representative TEM images of MSC-EVs from STR cultures. (B) Representative western blot images of MSC lysates and MSC-EVs samples. Detection of the housekeeping protein GAPDH, calnexin, synthenin and CD63 in MSC-EVs and corresponding WCL (i.e., cells) obtained from the STR operated under FB and FB/CP. Representative size distribution curves of EVs samples isolated from MSC-CM from FB (C) and FB/CP (D) cultures. WCL: whole cell lysates, STR: stirred tank reactor, FB: fed-batch, FB/CP: fed-batch combined with continuous medium perfusion, TEM: transmission electron microscopy.
The presence of EVs was also confirmed by Western blot analysis (Figure 2B). The EV protein markers synthenin and CD63 were successfully detected in EV samples, while calnexin, an endoplasmic reticulum protein, was present in cells but absent in EV samples, as expected. The size distribution of MSC-EVs was determined by NTA. In Figure 2C and D are depicted two representative size distribution curves for EVs isolated from MSC-CM from FB and FB/CP STR cultures, respectively. Isolated EVs showed an equivalent size distribution profile enriched in small EVs (<200nm) (Figure 2C and D). EVs mean and mode sizes were respectively (163±5.27) nm and (134±4.23) nm for FB cultures and (162±4.44) nm and (137±6.92) nm for FB/CP cultures Table 1(p>0.05). Moreover, the concentration of EVs in the MSC-CM from the STR cultures was also estimated by NTA. As presented in Table 1, average concentrations of (2.4±0.35)x1011 and (3.0±0.48)x1011 EVs/ml corresponding to (4.2±0.61)x1014 and (3.9±0.62)x1014EVs were estimated for EVs isolated from MSC-CM from STR operated under FB and FB/CP, respectively.
Table 1: MSC-EVs characteristics and concentration in MSC-CM.