High-Yield Production of Extracellular Vesicle Subpopulations with Constant Quality Using Batch-Refeed Cultures

The conventional manufacturing of extracellular vesicles (EVs) is characterized by low yields and batch-to-batch variability, hampering fundamental research on EVs and their practical applications. Perfusion operations have huge potential to address these limitations and increase the productivity and quality of EVs. In this study, perfusion cultures are simulated with batch-refeed systems and their productivity is compared with that achieved using batch cultures. It is shown that a shift from batch to batch-refeed system can increase the space-time yields of a target EV subpopulation characterized by CD81 and CD63 biomarkers by threefold. Moreover, it is demonstrated that the method facilitates the consistent production of the target EVs from cells maintained under constant conditions for 13 days. These results indicate that the use of perfusion cultures is a promising strategy to increase the manufacturing yield of EVs and control the production of specific EV subpopulations with constant quality attributes, thereby improving reproducibility.