Isolation and Characterization of Exosomes from Cultures of Tissue Explants and Cell Lines

Abstract Exosomes are specialized, nanometer‐sized extracellular vesicles of endosomal origin actively secreted into the extracellular space by a variety of cells under normal and pathological conditions. Exosomes have recently emerged as important intercellular communicators and modulators of diverse mechanisms and cellular responses. Characterization of their composition and function will open possibilities for new diagnostic methods and promising therapeutic approaches based on nanobiology. This unit provides a standard isolation procedure for purification of exosomes based on density gradient ultracentrifugation with sucrose. The process of isolating exosomes relies on obtaining proper source fluids/supernatants as well as qualitative and quantitative assessment of the isolated vesicles. The methodological procedures here can be divided in three parts: (1) pre‐isolation procedures aiming to obtain fluids containing exosomes, with a focus on protocols for organ explants and cell cultures; (2) a procedure for exosome isolation with several gradient alternatives; and (3) post‐isolation procedures for estimating the purity and yield of the exosomal fraction. © 2016 by John Wiley & Sons, Inc.