Figure 1: Ex-vivo expansion of human bone marrow-derived
mesenchymal stromal cells MSC(M) in a fully controlled stirred tank
reactor (STR) operated under fed-batch (FB) or FB combined with
continuous perfusion (FB/CP) operation mode. (A) STR working volume
during FB (orange line) and FB/CP (blue line) cultures. Growth curves in
terms of total cell number (B) and cells per mL (C) of MSC(M) expanded
on microcarriers in the STR operated under FB (orange line) or FB/CP
(blue line) operation mode (*P≤0.05). (C) Fold increase (FI) value in
total cell number estimated per day during FB (orange bars) and FB/CP
(blue bars) cultures. FI is the ratio between the number of cells
counted each day and the number of cells that adhered at day 1. Glucose
(light colour line) and lactate (dark colour line) concentrations
attained during time in culture for FB (E) and FB/CP (F) cultures. (G)
Representative images of microcarrier occupancy by MSC(M) stained with
DAPI throughout STR cultures. (F) Immunophenotypic analysis before
(light grey bars) and after expansion in the STR under FB (orange bars)
or FB/CP (blue bars) operation modes. 50×106 MSC(M)
from 3 donors were expanded on CellStart-coated plastic microcarriers
with xenogeneic/serum-free (X/S-free) StemPro culture medium. For FB
cultures, after 3 days with 600 mL of volume, fresh culture medium was
added at days 3, 6 and 7. For FB/CP cultures, from day 3 to day 5.5, 700
mL of fresh culture medium was added at a constant rate of 0.2 mL/min
and after reaching a volume of 1300 mL, the STR started to operate under
continuous perfusion at the same flow rate (0.22 day-1dilution rate) until day 11. Cell-containing microcarriers were
maintained inside the bioreactor, by using a Spin Filter mounted in the
agitation shaft. To maintain glucose concentration between 2-6 mM,
pulses of concentrated glucose were added to the STR daily (days 3-8) or
every 2 days (days 4-12) for FB and FB/CP cultures, respectively.
Results are presented as mean±standard error of the mean (n=3). STR:
stirred tank reactor, FB: fed-batch, FB/CP: fed-batch combined with
continuous medium perfusion.
In what concerns metabolic activity, the glucose and lactate
concentration profiles over time (Figure1E and F) show that the adopted
feeding schemes maintained glucose levels within the target range (2-6
mM), whereas lactate concentration, as expected, increases until
reaching a maximum of 18 mM at day 9 in FB cultures and remains
relatively constant below 6 mM in FB/CP cultures. To evaluate
qualitatively cell adhesion and proliferation of MSC immobilized on the
microcarriers during time in culture, DAPI staining was performed every
2 days for the different culture conditions tested (FB and FB/CP
operation modes). For both conditions, after the first 24h, a homogenous
cell distribution on the microcarriers was observed, followed by a
gradual increase in microcarrier occupancy by MSC throughout time in the
culture. At the end of culture, some cell-containing microcarrier
aggregation was observed, mainly for FB/CP cultures (Day 12) (Figure1G).
At the end of STR cultures, cells were detached from the microcarriers
and characterized by immunophenotypic analysis (Figure1H). MSC(M)
demonstrated high levels (>94%) of CD90 and CD73 and low
expression (<3%) of CD14, CD19, CD34, CD45 and HLA-DR
antigens before and after cell expansion in STR operated under both
feeding operation modes, with no major differences between them.
Concerning CD105, a decrease in its expression was observed after cell
expansion under stirred conditions [(99±0.51)% at day 0 to
(90±0.59)% and (87±1.5)% at day 8 and day 12, for FB and FB/CP,
respectively] (Figure1H).
3.2 Production of MSC-EVs in STR operated under FB or
FB/CP operation
mode
In the final two days of culture, for both FB and FB/CP cultures (day 7
and 11, respectively), no fresh culture medium was added, nor exhausted
medium removed from the STR, to concentrate the production of EVs. Only
concentrated glucose pulses were provided at days 8 and 12 for FB and
FB/CP cultures, respectively, to avoid glucose exhaustion.
After EV conditioning phase (last
2 days), the conditioned medium (MSC-CM) was collected, and the EVs were
successfully isolated and characterized according to “Materials and
Methods” section. EVs isolated from the MSC-CM of all STR cultures were
identified by TEM and Western blot. Representative TEM images depicted
in Figure 2A, show individual vesicles presenting a “cup-shaped
morphology” due to the adsorption of the EVs onto the hard surface and
drying.