Abstract P469: Scale-up And Characterization Of Therapeutic Mesenchymal Stromal Cell-derived Extracellular Vesicles

Introduction: Early studies have shown therapeutic benefits of mesenchymal stromal cells (MSCs) in cardioprotection due to their angiogenic, proliferative, anti-apoptotic and anti-inflammatory properties, which are now attributed to secreted factors such as extracellular vesicles (EVs). While MSC-EVs have shown promise in small animals for cardiovascular therapies, large animal studies are required to evaluate the therapeutic benefit of MSC-EVs for clinical translation. One of the biggest challenges for large animal studies is the need to generate clinically-relevant quality and quantity of EVs without batch-to-batch variations that could compromise efficacy. This study aims to explore three different cell culture methods (traditionally-used tissue culture plates (TCP), 3-D printed bioscaffolds in a perfusion system (P), and microcarriers in dynamic spinner flask conditions (M)) to scale-up the production of MSC-EVs across four different biological donors and rigorously investigate EV yield, size, shape, and content. Methods: MSCs were isolated from the iliac crest of four different Yucatan minipigs using heparinized syringes, and cells were expanded to passage four, at which point they were seeded onto the respective cell culture methods. EVs were collected from conditioned medium (CM) via differential ultracentrifugation. EV size, distribution, yield, and protein concentration were studied using Nanoparticle Tracking Analysis (NTA) and microBCA assays. Results: Both perfusion bioreactor and spinner flask systems enabled sustained maintenance of large numbers of cells. Across biological donors and fabrication methods, modes remained within 50-150 nm and were not statistically different. Microcarrier-based spinner flasks and perfusion bioreactor set-ups both improved EV yield, up to 6 times in efficiency. Ongoing research focuses on examining differences in EV content across biological donors using RNA-sequencing and proteomics.