Optimized exosome isolation protocol for cell culture supernatant and human plasma

Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, researchers from the QIMR Berghofer Medical Research Institute comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. They show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, their data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. The researchers find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, they provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity.

These results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. They have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. They believe this method will have extensive application in the growing field of extracellular vesicle research.

Exosomes isolated from plasma. (a) Size distribution profiles and EM images indicated all 3 protocols isolated particles of the correct size, size bar=200 nm. (b) Percentage recovery of particles in relation to unprocessed plasma showed no difference between EQ, ES and qEV. (c) Particle/protein ratio indicated that SEC using qEV columns significantly outperformed both EQ and ES. (d) Western blot analysis of 50 µg of protein indicated that all 3 methods were absent for Calnexin; however, Flotillin-1 could only be detected in the qEV lysate. The presence of non-exosomal contaminating proteins in EQ and ES samples was demonstrated with the abundance of albumin, which was almost absent in the qEV sample. n=3±SEM, **p<0.01, ***p<0.001. CL: cell lysate; EQ: ExoQuick™; ES: Exo-spin™; qEV: size exclusion columns.