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Abstract
Mesenchymal stromal cells (MSCs) are a promising therapeutic due to their immunomodulatory function, which has the potential to treat neuroinflammation associated with neurodegenerative diseases. This function is mediated, in part, by secreted extracellular vesicles (MSC-EVs). Despite established safety, MSC clinical translation has been generally unsuccessful due to inconsistent clinical outcomes resulting from functional heterogeneity. A major approach to mitigate functional heterogeneity is ‘priming’ MSCs with inflammatory signals, which enhances immunomodulatory function. However, comprehensive evaluation of priming conditions and their effects on MSC-EV function has not been performed. Furthermore, clinical translation of MSC-EV therapies requires significant manufacturing scale-up, yet few studies have investigated the effects of priming in bioreactors. MSC priming and EV manufacturing generally consists of MSC expansion, followed by a priming and MSC-EV release period, followed by MSC-EV harvesting, such that each batch of MSC-EVs incurs the full cost of expansion and concomitant heterogeneity. Continuous MSC-EV manufacturing strategies may be useful to increase the yield and consistency and reduce the cost of MSC-EV products. However, continuous MSC-EV manufacturing has not been investigated with priming. In this dissertation, we reviewed the potential of morphology as a critical quality attribute, then screened MSC morphological response to an array of priming signals and evaluated MSC-EV identity and bioactivity in response to priming in flasks and bioreactors. We discovered a novel bioreactor priming condition for producing MSC-EVs that modulates the immune response of microglia, which play a critical role in neuroinflammation, and gained mechanistic insight into this response via MSC-EV lipidomic profiling. Using the priming condition we discovered, we then characterized the MSC-EV response of 7 donors to a specific continuous manufacturing regimen. This work informs refinement of larger-scale MSC-EV manufacturing towards clinical translation of MSC-based therapies and represents a generalizable screening and manufacturing approach that could be applied to other cell therapy products and disease applications.