Equine mesenchymal stem cell-derived extracellular vesicle productivity but not overall yield is improved via 3-D culture with chemically defined media.

Mesenchymal stem cell (MSC) extracellular vesicles (EVs) have emerged as a biotherapeutic for osteoarthritis; however, manufacturing large quantities is not practical using traditional monolayer (2-D) culture. We aimed to examine the effects of 3-D and 2-D culture 2 types of media: Dulbecco modified Eagle medium and a commercially available medium (CM) on EV yield. Banked bone marrow-derived MSCs (BM-MSCs) from 6 healthy, young horses were used. 4 microcarriers (collagen-coated polystyrene, uncoated polystyrene, collagen-coated dextran, and uncoated dextran) were tested in static and bioreactor cultures, and the optimal microcarrier was chosen. The BM-MSCs were inoculated into a bioreactor with collagen-coated dextran microcarriers at 5,000 cells/cm2 or onto culture dishes at 4,000 cells/cm2 in either Dulbecco modified Eagle medium or CM media. Supernatants were obtained for metabolite and pH analysis. The BM-MSCs were expanded until confluent (2-D) or for 7 days (3-D) when the 48-hour EV collection period commenced using EV-depleted media. Extracellular vesicles were isolated and characterized via nanoparticle tracking analysis, Western blot, transmission electron microscopy, and protein quantification. The BM-MSCs were harvested, quantified, and immunophenotyped. The number of EVs isolated was not improved by 3-D culture or CM media, however, the CM 3-D condition improved the number of EVs produced per BM-MSC over the CM 2-D condition (mean ± SD: 306 ± 99 vs 37 ± 22, respectively). Glucose decreased and lactate and ammonium accumulated in 3-D culture. Surface markers of stemness exhibited reduced expression in 3-D culture. Optimization of our 3-D culture methods could improve BM-MSC expansion and thus EV yield.