Isolation and characterization of extracellular vesicles from tissues

Extracellular vesicles (EVs) are lipid bi-layered membrane structures released by all cells. Most EV studies have been performed using cell lines or body fluids, and studies on tissue-derived EVs are limited. Here we present a protocol to isolate EV subpopulations directly from tissues. This approach includes enzymatic treatment of dissociated tissues followed by differential ultracentrifugation and density separation. The isolated EV subpopulations were characterized by electron microscopy and RNA profiling. Additionally, their protein cargo was determined with mass spectrometry, Western blot, and ExoView™. The protocol requires extra steps compared to EV isolation from cell culture medium. Tissue-EV isolation can be performed in 22 hours, but a simplified version can be completed in 8 hours. Most experiments with the protocol have used human melanoma metastases, but the protocol can be applied to other cancer and non-cancer tissues. The procedure can be adopted by researchers experienced with cell culture and EV isolation.