Extracellular vesicles (EVs) have garnered significant interest in the biotechnology field due to their intrinsic therapeutic properties as well as their ability to serve as vehicles for bioactive cargo. EVs have also been shown to have significant clinical impact, specifically in vascularization. However, scalable biomanufacturing and limited potency of EVs in vivo remain critical bottlenecks for clinical translation. In this study, we utilized a 3D-printed scaffold-perfusion bioreactor system to assess the response of dynamic culture on extracellular vesicle production. We also investigated whether ethanol conditioning, which was previously shown to enhance vascularization bioactivity of endothelial cell (EC)-derived EVs produced in standard 2D culture conditions, could be employed successfully for the same purpose in a scalable production system. Particulary, we were interested in interrogating the impact of density, flow rate, and micro-environment on MSC EV production and vascular bioactivity using the bioreactor system.
Our results indicate that dynamic culture in a perfusion bioreactor significantly enhances EV production from human ECs. Moreover, the use of ethanol conditioning in conjunction with dynamic culture induces a pro-vascularization response from EC-derived EVs, which is correlated with increased EV levels of pro-angiogenic lncRNAs HOTAIR and MALAT1. This study represents one of the first reports of the integration of rationally-designed EV potency enhancement with scalable EV biomanufacturing.