Exosomes are small vesicles, approximately 30-100 nm in diameter, that transport various cargos, such as proteins and nucleic acids, between cells. It has been previously shown that exosomes can also transport viral proteins, such as the HTLV protein Tax, and viral RNAs, potentially contributing to disease pathogenesis. Therefore, it is important to understand their impact on recipient cells. Here, researchers from George Mason University describe methods of isolating and purifying exosomes from cell culture or tissue through ultracentrifugation, characterizing exosomes by surface biomarkers, and assays that evaluate the effect of exosomes on cells.
Purification of exosomes using ultracentrifugation and a gradient
The procedure utilizes 100–200 mL of cell supernatants at 2.5–5 × 106 cells/mL (depending on exosome concentration) that have been incubated in exosome-free media for 5 days. Cells are spun at 300 × g and 2000 × g to remove the cells and debris. The supernatants, which contain exosomes and other vesicles, are filtered using a 0.22 μm filter before being ultracentrifuged at 10,000 × g for 30 min to remove any microvesicles, apoptotic bodies, and other debris. This is followed by two 100,000 × g ultracentrifugation spins to pellet the exosomes, which are washed with DPBS without Ca2+/Mg2+ in between spins. The concentrated pellet can be resuspended in 50–200 μL and OptiPrep™, which utilizes an iodixanol gradient, can be run to separate particles by size. This will also separate most viruses from exosomes