Separation of blood-derived extracellular vesicles using droplet-based sorting 3166

Abstract Description Extracellular vesicles (EVs) are cell-derived, membrane-bound particles less than a micron in size and are valuable biomarkers and therapeutic agents. Due to their size and heterogeneity, EVs are difficult to characterize. Single Vesicle Flow Cytometry (vFC™) is a method to detect and analyze EVs. There is interest in droplet-based sorting to isolate EVs for further characterization. We present a proof-of-concept assay that purifies specific EV populations using a droplet-based sorter. A mixture of platelet- (PLT-EVs) and red blood cell-derived (RBC-EVs) EVs were analyzed using the vFC workflow, following MIFlowCyt-EV guidelines. A Cytek Aurora™ CS system with Enhanced Small Particle™ (ESP™) Detection Option was set up with a 70 μm nozzle. The sheath pressure was set at 30 psi to slow the sheath velocity and improve resolution. The threshold was set to membrane dye-positive EVs. CD41-positive PLT-EVs and CD235ab-positive RBC-EVs were sorted using multiway sort mode at an average sort rate of 500 events/second. The results of three independent experiments were similar, with an average efficiency and purity >98%. The average time to sort 1 million EVs was 25 minutes. This feasibility study demonstrates the purification of two EV populations using cell-of-origin surface markers and a high-sensitivity sorter. EV sorting may help address challenges in EV analysis and advance therapy and diagnostic development. Further experimentation will determine desired downstream workflows. Topic Categories Technological Innovations in Immunology (TECH)