Dual production of human mesenchymal stromal cells and derived extracellular vesicles in a dissolvable microcarrier-based stirred culture system

Cell therapies based on mesenchymal stromal cells (MSC) have gained an increasing therapeutic interest in the context of multiple disorders. Nonetheless, this field still faces important challenges, particularly concerning suitable manufacturing platforms. Here, dissolvable microcarriers were combined with xeno(geneic)-free culture medium to expand umbilical cord-derived Wharton's jelly MSC (MSC(WJ)) in a scalable stirred culture system. MSC(WJ) isolated from 3 donors were cultured at a starting seeding density of 1 × 106 cells per spinner flask (i.e. 2.8 × 103 cells per cm2 of microcarrier surface area). Taking advantage of an intermittent agitation regimen, we observed high adhesion rates to the microcarriers (over 90% at 24h) and achieved 15.8±0.7-fold expansion after 6 days of culture. Notably, the microcarriers used could be dissolved through a pectinase-based solution to recover the cell product, reducing the hurdles of downstream processing. MSC identity was validated by detecting the characteristic MSC immunophenotype, and by multilineage differentiation assays. Considering the growing interest in MSC-derived extracellular vesicles (EV), which are known to be mediators of the therapeutic features of MSC, this platform was also evaluated for EV production. Upon a 24h period of conditioning, secreted EV were isolated by ultrafiltration followed by anion-exchange chromatography and exhibited the typical cup-shaped morphology, small size distribution (162.6±30.2 nm) and expressed EV markers (CD63, CD9 and Syntenin-1). Taken together, we established a time-effective and robust scalable platform that complies with clinical-grade standards for the dual production of MSC(WJ) and their derived EV.