Guidelines for separation of natural extracellular vesicles

Abstract Extracellular vesicles (EVs) are found everywhere in different biofluids such as blood, saliva, urine, cerebrospinal fluid, and breast milk. They have sizes ranging from 20 to 400 nm and contain a wide range of cargo, including membrane proteins, periplasmic proteins, and nucleic acids. EVs possess the remarkable ability to diffuse, traverse the mucus layer, migrate directly to other tissues, and interact with various cells of the host immune system. Consequently, researchers have harnessed their potential as delivery carriers for novel immuno‐drugs or bioactive components. The separation of EVs from food can be considered a safe and effective method for harnessing their beneficial effects. In this guide, we present isolation protocols for natural EVs derived from plants, dairy products, cells, and microbes. This includes steps for raw material processing or culture as well as conditions for separation and purification. Moreover, these protocols can serve as a foundation for preparing and isolating engineered EVs. In summary, the utilization of natural EVs presents a vast array of opportunities in the fields of biotechnology, biomedicine, and food nutrition.