Engineering Human Bone Marrow-derived Mesenchymal Stromal Cell Aggregates for Enhanced Extracellular Vesicle Secretion in a Vertical-Wheel Bioreactor

Human mesenchymal stem/stromal cells (hMSCs) hold significant potential for regenerative medicine due to their anti-inflammatory and pro-angiogenic secretome. In particular, 3D hMSC aggregates secrete EVs that have enhanced anti-inflammatory and immunomodulatory properties. However, safety and efficacy concerns associated with stem cell therapy have led to the discovery of cell-free approaches utilizing hMSC-derived extracellular vesicles (EVs). Despite their therapeutic promise, the clinical application of hMSC-EVs is limited by low production yield. This study investigates the scalable generation of hMSC-EVs from 3D aggregates in a novel Vertical-Wheel Bioreactor (VWBR) through shear stress-mediated biochemical cues, promoting cargo expression relevant to nerve regeneration and neuropathic treatment. Bone marrow-derived hMSCs were cultured as 3D aggregates in VWBRs and exposed to two different culture media—aMEM/FBS (serum-containing) and DMEM/F12/B27 (serum-free)—under three agitation speeds (25, 40, and 64 rpm). Metabolite and gene analyses were performed to assess EV biogenesis, focusing on ESCRT machinery markers. Results showed that hMSCs cultured in VWBR exhibited higher expression of EV biogenesis genes and glycolytic genes compared to static culture. aMEM/FBS (serum-containing) condition was more robust than DMEM/F12/B27 (serum-free) condition. EV yield (EV number per cell) increased by 3-10 fold (in serum-containing medium) in VWBR compared to static culture, with size of 120-180 nm and EV marker expression. microRNA-sequencing of the EVs shows the upregulation of miR-29a-3p, miR-451a, miR-224-5p, miR-16-5p, miR-133a-3p, and miR-143-3p, etc., reflecting a combination that enhances EV biogenesis, promotes metabolic reprogramming, and supports immunomodulatory behavior characteristics. Functional assays demonstrated that the generated EVs effectively modulated Schwann cell responses under neural inflammation, supporting their therapeutic potential. These findings highlight the role of VWBR-driven hydrodynamics in promoting EV production from 3D hMSC aggregates. This study advances the knowledge of dynamic aggregation and metabolic influence on 3D hMSC-EV production and fundamentally scale up the preconditioned techniques used to promote hMSC aggregation and the consequent EV secretion. Overall design: Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) were cultured in regular 2D or 3D conditions. EVs were collected and total RNA was isolated for sequencing.