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.