Protocols for isolating exosomes frequently include differential ultracentrifugation with a density gradient. What benefits does this method offer?

Differential ultracentrifugation has long been considered the 'gold standard' for isolating exosomes because it can reliably provide pure exosomes for study.1,2 A variation on this method using a 30% sucrose cushion eliminates even more contaminants—such as proteins not associated with exosomes, or large protein aggregates—which are sedimented by centrifugation but don’t float on a sucrose gradient. More recently, density gradient centrifugation with iodixanol has outperformed standard ultracentrifugation of CCM in terms of purity for downstream omics profiling.3,4 Density gradients enable efficient separation of vesicles according to their density5, thus providing a “cleaner” population of exosomes.2

[1] Théry C, Amigorena S, Clayton A and Raposo G. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. Chapter 3; Unit 3.22: (2006).

[2] Momen-Heravi F, Balaj L, et al. Current methods for the isolation of extracellular vesicles. Biol Chem. 394;1253-62: (2013). doi: 10.1515/hsz-2013-0141.

[3] Van Deun J, Mestdagh P, Sormunen R, et al. The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J Extracell Vesicles. 3; 24858: (2014). doi: org/10.3402/jev.v3.24858.

[4] Lobb RJ, Becker M, Wen SW, et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles. 4; 27031: (2015). doi: org/10.3402/jev.v4.27031.

[5] Witwer KW, Buzás EI, Bemis LT, et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles. 2; 20360: (2013). doi: org/10.3402/jev.v2i0.20360.