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.