High-Purity Enrichment of Extracellular Vesicles from Diverse Sources by Conventional and Image-Based Fluorescence Activated Cell Sorters for Robust Downstream Applications

Separating and enriching specific extracellular vesicle (EV) subpopulations from the broader EV pool present in tissues and blood is crucial for understanding their role in physiological and pathological conditions. However, high-purity enrichment of specific EV-subpopulations remains challenging due to the lack of suitable techniques. Initial studies have shown that Fluorescence-Activated Cell Sorting (FACS) has great potential for enriching EV subpopulations, despite the technical challenges posed by their small size. Yet, existing protocols have been inconsistent, and proper validation using state-of-the-art sorters has been inadequate. Here, we introduce an EV sorting workflow that overcomes technical challenges and allows for the analysis of EVs from various species, tissue sources and cell culture. We used two fluorescence cell sorters, the BD FACSAria Fusion and the BD FACSDiscover S8, to sort EVs with different fluorescent labels. The successful sorting of EVs was validated using high-sensitivity imaging flow cytometry, transmission electron microscopy, and liquid chromatography tandem mass spectrometry. We defined the optimal parameters for nozzle sizes, flow rates, sample dilutions, and sorting modes, enabling the enrichment of EV populations of interest to nearby 100% purity, including low-frequency EV populations of under 10%, while preserving compatibility with downstream analyses. The workflow presented here provides a powerful tool for both, basic science and translational applications.