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Abstract
Mesenchymal stromal cells (MSCs) offer significant therapeutic potential for neurodegenerative diseases, owing to their intrinsic immunomodulatory properties. However, several risks associated with MSCs, such as heterogeneity, thrombosis, uncontrolled differentiation, and the need for invasive application procedures, pose significant challenges to their use in clinical settings. Fortunately, MSCs secrete extracellular vesicles (MSC-EVs) that carry similar immunomodulatory potential as their parent cells. MSC-EVs are heterogeneous, non-replicating particles characterized by a lipid bilayer, and they are shed from the MSC plasma membrane. The immunomodulatory effects of MSC-EVs are driven by the bioactive cargo they carry, including proteins, RNAs, DNAs, enzymes, and other molecules. MSC-EVs offer advantages over their parent cells by avoiding many of the risks associated with MSCs, including the challenges posed by living cells. Additionally, MSC-EVs can be easily taken up by other cells and can cross the blood-brain barrier, making them an attractive therapeutic option for neurodegenerative diseases. However, challenges remain in the standardization of MSC-EV manufacturing processes and bioactivity assessment, limiting their clinical application. Thisdissertation aims to address these challenges by examining how different manufacturing conditions affect MSC-EV characteristics, with the goal of identifying standardized methods for their production. We first investigated how different priming conditions influenced the generation of bioactive MSC-EVs, focusing on their potential use in neurodegenerative disease applications. To do this, we developed a human brain pericyte morphological assay to screen MSC-EVs. Using this assay, we identified optimal priming conditions (e.g., cytokines and oxygen levels) and culture platforms (flasks versus bioreactors) for producing immunomodulatory MSC-EVs. Additionally, we compared the physical and functional characteristics of MSC-EVs isolated using tangential flow filtration and ultracentrifugation from four different MSC lines derived from two tissue sources (bone marrow and adipose tissue). While we observed differences in the size and concentration of MSC-EV populations and their cargo, the functional properties (e.g., pericyte morphology, macrophage IL-6 secretion) of the vesicles were largely unaffected by the isolation method used. Overall, this work provides key insights into the manufacturing of MSC-EVs and highlights important considerations for developing standardized production methods for their use in neurodegenerative disease therapies.