Engineering Extracellular Vesicle Production Environments Towards Scalability and Enhanced Therapeutic Potency

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Impaired wound healing remains a critical issue in numerous life threatening diseases, affecting more than 40 million patients worldwide with an annual cost exceeding $100 billion. Despite significant advancements in the field of regenerative medicine, a clinically viable molecular or cell based approach to wound healing remains to be established. Recently, cell-derived extracellular vesicles (EVs), which are nanoscopic lipid-bilayer membrane vesicles, have emerged as a new class of therapeutics that autonomously enhance the wound healing process through the transfer of bioactive cargo including protein and RNA to recipient cells. However, EVs face critical limitations to clinical translation, especially their low efficacy and the lack of standardized methods for large-scale exosome biomanufacturing. The goal of this

project is to address these limitations through a systematic investigation of the impact of cell culture and mechanical microenvironment on EV secretion and bioactivity. We showed that low seeding density caused increase in EV output per cell and EVs produced from mesenchymal stem cells (MSCs) at higher culture passages, had significantly lower bioactivity in an in vitro wound healing assay. Next, we adapted the first use of a perfusion bioreactor system with 3D printing scaffolds to culture mesenchymal stem cells (MSCs) and endothelial cells (ECs) for large-scale EV production. Subjecting ECs and MSCs to physiological shear stresses in the bioreactor resulted in significantly higher EV release compared to tissue culture flask without diminishing their bioactivity. Additionally, stimulation of ECs with ethanol induced provascularization bioactivity of EC-derived EVs, which was correlated with increased levels of angiogenic lncRNAs HOTAIR and MALAT1 in EVs. Further, MSC-derived EV production and bioactivity was maximized through systematic exposure to increasing shear stress and cyclic stretch. Through this study, we outlined specific culture environment that is suitable for increasing both potency as well as EV biomanufacturing, a first in the field. In this project, we integrated fundamental knowledge of how the cellular microenvironment and phenotype influences EV generation and bioactivity into a rationally designed biomanufacturing platform for therapeutic EV production towards wound healing and open a novel avenue for developing clinically relevant EV-based therapies.