Optimized operation of a controlled stirred tank reactor system for the
production of mesenchymal stromal cells and their extracellular vesicles
Abstract
The therapeutic effects of human mesenchymal stromal cells (MSC) have
been attributed mostly to their paracrine activity, exerted through
small-secreted extracellular vesicles (EVs) rather than their
engraftment into injured tissues. Currently, the production of
MSC-derived EVs (MSC-EVs) is performed in laborious static culture
systems with limited manufacturing capacity using serum-containing
media. In this work, a serum-/xenogeneic-free microcarrier-based culture
system was successfully established for bone marrow-derived MSC
cultivation and MSC-EV production using a 2 L-scale controlled stirred
tank reactor (STR) operated under fed-batch (FB) or fed-batch combined
with continuous perfusion (FB/CP). Overall, maximal cell numbers of
(3.0±0.12)×10 8 and (5.3±0.32)×10 8
were attained at days 8 and 12 for FB and FB/CP cultures, respectively,
and MSC(M) expanded under both conditions retained their
immunophenotype. MSC-EVs were identified in the conditioned medium
collected from all STR cultures by TEM, and EV protein markers were
successfully identified by WB analysis. Overall, no significant
differences were observed between EVs isolated from MSC expanded in STR
operated under the two feeding approaches. EV mean sizes of 163±5.27 nm
and 162±4.44 nm (P>0.05) and concentrations of
(2.4±0.35)x10 11 EVs/mL and (3.0±0.48)x10
11 EVs/mL (P>0.05) were estimated by
nanoparticle tracking analysis for FB and FB/CP cultures, respectively.
The STR-based platform optimized herein represents a major contribution
towards the development of human MSC- and MSC-EV-based products as
promising therapeutic agents for Regenerative Medicine settings.