Advanced 3D dynamic culture system with transforming growth factor-β3 enhances production of potent extracellular vesicles with modified protein cargoes via upregulation of TGF-β signaling.

INTRODUCTION:Mesenchymal stromal cells (MSCs) release extracellular vesicles (MSC-EVs) containing various cargoes. Although MSC-EVs show significant therapeutic effects, the low production of EVs in MSCs hinders MSC-EV-mediated therapeutic development. OBJECTIVES:Here, we developed an advanced three-dimensional (a3D) dynamic culture technique with exogenous transforming growth factor beta-3 (TGF-β3) treatment (T-a3D) to produce potent MSC-EVs. METHODS:Our system enabled preparation of a highly concentrated EV-containing medium for efficient EV isolation and purification with higher yield and efficacy. RESULTS:MSC spheroids in T-a3D system (T-a3D spheroids) showed high expression of CD9 and TGF-β3, which was dependent on TGF-β signaling. Treatment with EVs produced under T-a3D conditions (T-a3D-EVs) led to significantly improved migration of dermal fibroblasts and wound closure in an excisional wound model. The relative total efficacy (relative yield of single-batch EVs (10-11-fold) × relative regeneration effect of EVs (2-3-fold)) of T-a3D-EVs was approximately up to 33-fold higher than that of 2D-EVs. Importantly the quantitative proteomic analyses of the T-a3D spheroids and T-a3D-EVs supported the improved EV production as well as the therapeutic potency of T-a3D-EVs. CONCLUSION:TGF-β signalling differentially regulated by fluid shear stress produced in our system and exogenous TGF-β3 addition was confirmed to play an important role in the enhanced production of EVs with modified protein cargoes. We suggest that the T-a3D system leads to the efficient production of MSC-EVs with high potential in therapies and clinical development.