The potential of exosomes as biomarker resources for diagnostics, prognostics and even for therapeutics is an area of intense research. Despite the various approaches available, there is no consensus with respect to the best methodology for isolating exosomes and to provide substantial yields with reliable quality. Differential centrifugation is the most commonly used method but it is time-consuming and requires large sample volumes, thus alternative methods are urgently needed.
In this study researchers from the University of Aveiro compared two precipitation-based methods and one column-based approach for exosome isolation from distinct biofluids (serum, plasma and cerebrospinal fluid). Exosome characterization included morphological analyses, determination of particle concentration, stability and exosome preparations’ purity, using different complementary approaches such as Nanoparticle Tracking Analysis, Electrophoretic Light Scattering, Transmission Electron Microscopy, EXOCET colorimetric assay, protein quantification methods and western blotting. The three commercial kits tested successfully isolated exosomes from the biofluids under study, although ExoS showed the best performance in terms of exosome yield and purity. Data shows that methods other than differential centrifugation can be applied to quickly and efficiently isolate exosomes from reduced biofluid volumes. The possibility to use small volumes is fundamental in the context of translational and clinical research, thus the results here presented contribute significantly in this respect.
Exosome isolation workflow
Serum and plasma-derived exosomes were isolated using the precipitation-based reagents TEI and ExoQ; and the column-based method ExoS. Pooled exosomes were characterized at different levels and using different methodologies. Exosome extraction from CSF was also performed using equivalent methods. *Serum and plasma-derived exosome preparations obtained using ExoS were at this step ressuspended in higher PBS volumes due to exosome retention in the column.