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. • Protocol for purifying human plasma EVs without ultracentrifugation • Instructions for EV purification using TFF, PEG precipitation, and chromatography • Guidance on multimodal and heparin affinity chromatography for plasma EV cleanup • EV characterization by NTA, Western blot, Coomassie stain, fusion assay, and dSTORM Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.