Protocol for the isolation and characterization of extracellular vesicles from human mesenchymal stromal cells

Here, we present a protocol for isolating extracellular vesicles (EVs) from the conditioned medium of human mesenchymal stromal cells (MSCs) and characterizing them using multiple modalities. We describe steps for enriching EVs by ultrafiltration and size-exclusion chromatography. We then detail procedures for imaging flow cytometry, transmission electron microscopy, nanoparticle tracking analysis, and the extraction of EV protein and RNA content. This workflow enables an integrated assessment of MSC-derived EVs to inform their use in diagnostic or therapeutic applications.