Protocol for large-scale, high-yield, high-purity extracellular vesicle purification from human plasma.

Extracellular vesicles (EVs) hold promise as biomarkers and drug delivery vehicles; however, their broader use is limited by the lack of effective and scalable purification methods. We present a protocol for purifying EVs from human plasma without ultracentrifugation. The workflow incorporates tangential flow filtration (TFF) for volume reduction, polyethylene glycol (PEG)-based precipitation for enrichment, nuclease treatment to remove extravesicular nucleic acids, and multimodal and affinity chromatography for contaminant depletion. We further describe procedures for biochemical, biophysical, and functional EV characterization.