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Purpose: 3D cell culture and hypoxia have been demonstrated to increase the therapeutic effects of mesenchymal stem/stromal cells (MSCs)-derived extracellular vesicles (EVs). In this study, a process for the production of MSC-EVs in a novel 3D bioreactor system under normoxic and hypoxic conditions was established and the resulting EVs were characterized.
Methods: Human adipose-derived MSCs were seeded and cultured on a 3D membrane in the VITVO® bioreactor system for 7 days. Afterwards, MSC-EVs were isolated and characterized via fluorescence nanoparticle tracking analysis, flow cytometry with staining against annexin V (Anx5) as a marker for EVs exposing phosphatidylserine, as well as CD73 and CD90 as MSC surface markers.
Results: Cultivation of MSC in the VITVO® bioreactor system demonstrated a higher concentration of MSC-EVs from the 3D bioreactor (9.1 × 10 ± 1.5 × 10 and 9.7 × 10 ± 3.1 × 10 particles/mL) compared to static 2D culture (4.2 × 10 ± 7.5 × 10 and 3.9 × 10 ± 3.0 × 10 particles/mL) under normoxic and hypoxic conditions, respectively. Also, the particle-to-protein ratio as a measure for the purity of EVs increased from 3.3 × 10 ± 1.1 × 10 particles/µg protein in 2D to 1.6 × 10 ± 8.3 × 10 particles/µg protein in 3D. Total MSC-EVs as well as CD73CD90 MSC-EVs were elevated in 2D normoxic conditions. The EV concentration and size did not differ significantly between normoxic and hypoxic conditions.
Conclusion: The production of MSC-EVs in a 3D bioreactor system under hypoxic conditions resulted in increased EV concentration and purity. This system could be especially useful in screening culture conditions for the production of 3D-derived MSC-EVs.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10902030 | PMC |
| http://dx.doi.org/10.1007/s10529-024-03465-4 | DOI Listing |
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