Validation of Extracellular Vesicles in FACTORFIVEs EXOFECTA LyoBeads
Stachura, D. L.; Rueb, K. F.; Hadley, D.; Bozkurt, B.; Carney, R.; Aylworth, J.
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ObjectiveThe objective of this project was to determine if extracellular vesicles (EVs) were present in FACTORFIVEs EXOFECTA LyoBeads to validate flash freezing and subsequent lyophilization as an effective EV preservation method. SummaryEVs were isolated from resuspended EXOFECTA LyoBeads using differential ultracentrifugation and subjected to further characterization techniques such as nanoparticle tracking analysis (NTA), single particle flow cytometry, and cryo-electron microscopy (cryo-EM). First, NTA revealed particles with size distributions and concentrations consistent with the expected characteristics of EVs, indicating successful isolation from samples. Secondly, flow cytometry was performed using antibodies targeting the canonical EV tetraspanins CD9, CD63, and CD81. Detection of these markers supported the presence of enriched EVs in the samples. Finally, cryo-EM was performed to confirm the existence of vesicular bodies and their morphology. Visualization of the samples revealed membrane-enclosed particles with a visible lipid bilayer, consistent with EVs. Overall, the results support EVs were in EXOFECTA LyoBeads, indicating EV retention in the final product after FACTORFIVEs method of flash freezing, stabilizing, and lyophilization.
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