Mechanically Regulated Production for Improved Yield and Therapeutic Potency of Mesenchymal Stem Cell Extracellular Vesicles

Abstract

Mesenchymal stem cells (MSCs) are among the most extensively studied cell types for regenerative therapies due to their intrinsic pro-angiogenic and anti-inflammatory properties. However, MSC-based therapies face persistent challenges including risks of tumorigenicity and spontaneous differentiation. Extracellular vesicles (EVs)- cell-secreted nanovesicles that shuttle bioactive cargo reflective of their parent cells- offer a promising alternative by recapitulating the therapeutic benefits of MSCs while avoiding cell-associated risks. Accordingly, the therapeutic relevance of MSC EVs extends across numerous indications including cardiovascular disease, inflammatory diseases, cancer, and wound healing. Despite their potential, the clinical development of MSC EVs remains limited by donor variability, low innate potency, and lack of rationally designed scalable manufacturing approaches. Moreover, conventional strategies to address low potency (e.g. genetic engineering, exogenous cargo loading) are generally incompatible with large-scale biomanufacturing and substantially increase complexity and cost. Previous studies have shown that bone marrow-derived MSCs (BMMSCs) respond favorably to mechanical cues, yet both the cells and their EVs are hindered by donor variability and early senescence. To overcome these barriers, we leveraged induced pluripotent stem cell-derived MSCs (iMSCs) as a renewable and consistent EV source, and harnessed mechanical cues- specifically substrate stiffness and cellular confinement- and bioreactor-based cell culture to improve EV production and potency. We demonstrated that culturing iMSCs on soft substrates enhances both EV yield and angiogenic activity in a stiffness-dependent manner, paralleling BMMSC responses. Additionally, we showed that high degrees of cell confinement promote superior angiogenic function of the secreted iMSC EVs. Building on these findings, we developed and optimized a 3D-printed perfusion bioreactor incorporating cell confinement, achieving a 67-fold increase in iMSC EV production compared to traditional flask culture while preserving the enhanced bioactivity conferred by confinement alone. Importantly, treatment with the bioreactor-generated EVs accelerated wound healing in a diabetic mouse model compared to flask-generated EVs and the vehicle control. Overall, this work establishes mechanobiology-based EV manufacturing platforms that address key translational bottlenecks in MSC EV therapeutic development. By integrating renewable cell sources with scalable, potency-enhancing biomanufacturing strategies, these findings advance the path toward consistent, large-scale production of MSC EVs suitable for clinical translation.