Evaluating Extracellular Vesicle Production Scalability with Confinement and Flow

Abstract

Mesenchymal stem cell (iMSC) Extracellular Vesicles (EVs) are nano-sized particles naturally secreted by cells, which are good therapeutic delivery molecules as they have beneficial effects on regenerative processes, are biocompatible, and can naturally cross the cytoplasmic membrane. Although these EVs have such therapeutic benefits, they lack potency, increasing regulatory burden and making them less effective in clinical practice. Therefore, my goal is to improve the biomanufacturing of EVs with a focus on using confinement to increase EV potency and ensuring EV bioactivity is retained when bioreactors are utilized. EVs already have pro-angiogenic properties, but I want to increase this property by producing EVs from iMSCs grown at different confinements on micropillar devices. Using tube formation assays, I confirmed an increase in cell confinement—5 micron distance was optimal—resulted in greater bioactivity in EVs. These experiments were performed in a static culture; however, for EV production to be sustainable when scaled up, large-scale bioreactors are required. These bioreactors introduce a new variable for testing: media flow. Using tube formation assays, I confirmed EV bioactivity was retained with this new variable by testing EVs produced from iMSCs grown at 5 microns apart in a bioreactor with media flow and on a static culture with no flow. The potency was measured by the relative number of tubes formed by HUVECs when introduced to the two experimental groups and compared using a treatment control. The next step for this research project is to optimize the flow rate of the bioreactor to further increase EV bioactivity.