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Extracellular vesicles (EVs) are implicated as promising therapeutics and drug delivery vehicles in various diseases. However, successful clinical translation will depend on development of scalable biomanufacturing approaches, especially due to the documented low levels of intrinsic EV-associated cargo that may necessitate repeated doses to achieve clinical benefit in certain applications. Thus, here we assessed effects of a 3D-printed scaffold-perfusion bioreactor system on the production and bioactivity of EVs secreted from bone marrow-derived mesenchymal stem cells (MSCs), a cell type heavily implicated in generating EVs with therapeutic potential. Our results indicate that perfusion bioreactor culture results in an ~40-80-fold increase, depending on measurement method, in MSC EV production compared to conventional cell culture. Additionally, we demonstrated that MSC EVs generated using the bioreactor system significantly improved wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Overall, this study establishes a promising solution to major EV translational issues (i.e., scalability and low potency) with potential for adaptation to various EV-based therapeutics and capacity for improvement alongside the continuous advancements in 3D-printing technologies.

Chronic wounds remain a substantial source of morbidity worldwide. An emergent approach that may be well suited to inducing the complex, multicellular processes such as angiogenesis that are required for wound repair is the use of extracellular vesicles (EVs). EVs contain a wide variety of proteins and nucleic acids that enable multifactorial signaling. Here, we leverage the capability of EVs to be engineered via producer cell modification to investigate the therapeutic potential of EVs from mesenchymal stem/stromal cells (MSCs) transfected to overexpress long non-coding RNA HOX transcript antisense RNA (HOTAIR). HOTAIR was previously shown by our group to be critical in mediating angiogenic effects of endothelial cell EVs, and MSCs were chosen as EV producer cells for this study due to their widely reported intrinsic angiogenic properties. The results indicate that MSCs overexpressing HOTAIR (HOTAIR-MSCs) produce EVs with increased HOTAIR content that promote angiogenesis and wound healing in a diabetic (db/db) mice. Further, endothelial cells exposed to HOTAIR-MSC EVs exhibit increased HOTAIR content correlated with upregulation of the angiogenic protein vascular endothelial growth factor (VEGF). Thus, this study supports EV-mediated HOTAIR delivery as a strategy for further exploration towards healing of chronic wounds.