Enhanced extracellular vesicle production and ethanol-mediated vascularization bioactivity via a 3D-printed scaffold-perfusion bioreactor system.

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. However, the lack of an established biomanufacturing platform 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 from endothelial cells (ECs). We also investigated whether ethanol conditioning, which was previously shown to enhance vascularization bioactivity of EC-derived EVs produced in standard 2D culture conditions, could be employed successfully for the same purpose in a 3D production 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 pro-vascularization bioactivity of EC-derived EVs that is correlated with increased EV levels of pro-angiogenic lncRNAs HOTAIR and MALAT1. Thus, this study represents one of the first reports of rationally-designed EV potency enhancement that is conserved between static 2D and dynamic 3D EV production systems, increasing the potential for scalable biomanufacturing of therapeutic EC-derived EVs for a variety of applications. STATEMENT OF SIGNIFICANCE: Extracellular vesicles (EVs) have substantial therapeutic potential in a variety of applications. However, translation of EV-based therapies may be hindered by biomanufacturing challenges. EV production to date has predominantly involved the use of tissue culture flasks. Here, we report, for the first time, the use of a tubular perfusion bioreactor system with an integrated 3D-printed biomaterial scaffold for EV production from human endothelial cells. This system increases EV yield by over 100-fold compared to conventional tissue culture systems. Further, we show that an ethanol-conditioning approach that our group previously developed in 2D culture for enhancing EV potency is compatible with this new system. Thus, potency enhancement of EVs for vascularization applications is possible even with significantly increased production rate.