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

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.