Isolation of Exosomes and Microvesicles from Cell Culture Systems to Study Prion Transmission

Extracellular vesicles (EVs) are composed of microvesicles and exosomes. Exosomes are small membrane vesicles (40-120 nm sized) of endosomal origin released in the extracellular medium from cells when multivesicular bodies fuse with the plasma membrane, whereas microvesicles (i.e., shedding vesicles, 100 nm to 1 μm sized) bud from the plasma membrane. Exosomes and microvesicles carry functional proteins and nucleic acids (especially mRNAs and microRNAs) that can be transferred to surrounding cells and tissues and can impact multiple dimensions of the cellular life. Most of the cells, if not all, from neuronal to immune cells, release exosomes and microvesicles in the extracellular medium, and all biological fluids including blood (serum/plasma), urine, cerebrospinal fluid, and saliva contain EVs.Prion-infected cultured cells are known to secrete infectivity into their environment. We characterized this cell-free form of prions and showed that infectivity was associated with exosomes. Since exosomes are produced by a variety of cells, including cells that actively accumulate prions, they could be a vehicle for infectivity in body fluids and could participate to the dissemination of prions in the organism. In addition, such infectious exosomes also represent a natural, simple, biological material to get key information on the abnormal PrP forms associated with infectivity.In this chapter, we describe first a method that allows exosomes and microvesicles isolation from prion-infected cell cultures and in a second time the strategies to characterize the prions containing exosomes and their ability to disseminate the prion agent.