Perfusion Bioreactor Culture Incorporating Mechanical Confinement Enhances Mesenchymal Stem Cell Extracellular Vesicle Production and Wound Healing Potential

ABSTRACT Mesenchymal stem cell extracellular vesicles (MSC EVs) have been widely studied for regenerative medicine and tissue repair applications. However, clinical translation of EV therapeutics has been hampered by low potency and a lack of scalable production strategies. This work aims to develop a novel approach that exploits the mechanosensitivity of MSCs to enhance EV potency in the context of enhanced production via bioreactor culture. MSCs are well known to respond to mechanical stimuli such as substrate stiffness and shear stress, and here it is shown that exposing MSCs to another mechanoregulatory parameter, confinement, enhances the pro‐angiogenic bioactivity of their EVs. This is consistent across both donor‐derived primary MSCs and induced pluripotent stem cell‐derived MSCs (iMSCs). The development of a 3D‐printed perfusion bioreactor system that enables culture of confined MSCs under flow is also detailed here, resulting in a 67‐fold increase in EV production compared to flask culture. iMSC EVs obtained downstream of this confinement‐bioreactor culture induce greater vascularization and generally improve wound healing in a diabetic mouse model compared to iMSC EVs from conventional tissue culture. Overall, this work establishes the development of a scalable, bioreactor‐based iMSC EV production platform that provides a solution to major translational bottlenecks of therapeutic EVs.