Are reagent kits and/or microfiltration effective alternatives to differential ultracentrifugation for isolating exosomes?

FAQs Research

Generally speaking, ultracentrifugation is the 'gold standard' for exosome isolation, while other methods are newer and less widely adopted. Isolation reagent kits generally rely on immunophenotype-based enrichment of the exosome sample, which requires antibodies to bind with surface proteins on the exosome.2 The antibodies are usually bound to beads, which enables rapid, density-based isolation of bead-bound exosomes. These kits offer the advantages of delivering results much faster while requiring no expensive equipment or technical knowledge, although many kit manufacturers have not disclosed or validated their mode-of-action.

Which method is most effective for purifying exosomes? Ultimately, the “best” method for exosome isolation is the one that works best with your samples, in your laboratory and in your hands. There is no one-size-fits-all answer.3 

Case in point: for serum exosomal miRNA profiling, a commercially available isolation reagent kit can isolate exosomes as effectively as ultracentrifugation. Other researchers have reported conflicting results when using isolation reagent kits for different protocols and downstream analysis.1 In one study, more exosomes were isolated using ultracentrifugation with a reagent kit, but isolation of higher-quality exosomes—with intact morphological structures—was achieved with ultracentrifugation with a density gradient (and no reagent kit).

Similarly, some researchers report no significant difference using ultrafiltration with differential ultracentrifugation,4 while others describe how ultrafiltration resulted in a higher recovery of particles <100 nm (indicating exosomes) compared to using ultracentrifugation alone.4 As with many aspects of exosome research, more study is needed.

[1] 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.

[2] Rekker K, Saare M, Roost AM, et al. Comparison of serum exosome isolation methods for microRNA profiling. Clin Biochem. 47(1-2); 135–138: (2014). doi: org/10.1016/j.clinbiochem.2013.10.020.

[3] Yamada T, Inoshima Y, et al. Comparison of Methods for Isolating Exosomes from Bovine Milk. J. Vet. Med. Sci. 74(11); 1523–1525: (2012). doi: 10.1292/jvms.12-0032.

[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.