Optimization of Mammalian Cell Culture for Extracellular Vesicle Production, Isolation, and Characterization using Nano Flow Cytometry for Future Applications in Diagnostics and Nanoparticle Therapeutics

Extracellular vesicles (EVs) are lipid bilayer-bound nanoparticles secreted by cells for use in intercellular trafficking and communication. EVs have shown promise for use in diagnostics and nanoparticle therapeutics, but in vitro studies have been hindered by suboptimal and inconsistent EV handling, isolation, and analysis techniques. This study aimed to optimize mammalian cell culture techniques for EV yield and purity, utilize nano flow cytometry (nFCM) to identify optimal EV isolation procedures, and assess the applicability of these methods across clinically relevant mammalian cell lines. The results of this study suggest that cells cultured in serum-free media for 20 hours prior to EV isolation with ultracentrifugation result in EV samples of high yield and purity, with minimal impacts on donor cell viability. Further, particle counts and lipid purity data obtained using precise nFCM analysis suggests the applicability of these techniques to human glioblastoma and mesenchymal stem cell lines. Future work utilizing these methods may aid progress towards improved EV characterization, more precise EV diagnostic applications, and effective therapeutic EV interventions.