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Optimized exosome isolation protocol for cell culture supernatant and human plasma.

Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A - J Extracell Vesicles (2015)

Bottom Line: Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield.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, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity.

View Article: PubMed Central - PubMed

Affiliation: Tumour Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, Herston, QLD, Australia.

ABSTRACT
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, we 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. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our 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. We 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, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our 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. We 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. We believe this method will have extensive application in the growing field of extracellular vesicle research.

No MeSH data available.


Related in: MedlinePlus

The NSCLC SK-MES-1 cell line produces exosomes that can be isolated with ultrafiltration of CCM. (a) 5 µg of protein was used for Western blot analysis of isolated exosomes. The presence of canonical exosome proteins, and the absence of Calnexin demonstrates a pure exosome preparation. (b) TRPS analysis demonstrates a size distribution of particles consistent with the size range of exosomes. (c) EM image of exosomes demonstrates cup-shape morphology, size bar=200 nm. CL: cell lysate; E: exosome lysate.
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Figure 0001: The NSCLC SK-MES-1 cell line produces exosomes that can be isolated with ultrafiltration of CCM. (a) 5 µg of protein was used for Western blot analysis of isolated exosomes. The presence of canonical exosome proteins, and the absence of Calnexin demonstrates a pure exosome preparation. (b) TRPS analysis demonstrates a size distribution of particles consistent with the size range of exosomes. (c) EM image of exosomes demonstrates cup-shape morphology, size bar=200 nm. CL: cell lysate; E: exosome lysate.

Mentions: The identification of particles as exosomes relies on various criteria. Exosomes range in size from 50 to 100 nm (8), they display a cup-shaped morphology by EM, and proteomic analysis reveals the presence of common proteins (9–11). Figure 1a demonstrates the presence of canonical exosome proteins and the absence of the endoplasmic reticulum protein Calnexin from exosomes isolated using the ultrafiltration protocol (Supplementary Fig. 1). Furthermore, isolated particles exhibit expected morphology and size profiles consistent with pure exosome preparations (Fig. 1b and c).


Optimized exosome isolation protocol for cell culture supernatant and human plasma.

Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A - J Extracell Vesicles (2015)

The NSCLC SK-MES-1 cell line produces exosomes that can be isolated with ultrafiltration of CCM. (a) 5 µg of protein was used for Western blot analysis of isolated exosomes. The presence of canonical exosome proteins, and the absence of Calnexin demonstrates a pure exosome preparation. (b) TRPS analysis demonstrates a size distribution of particles consistent with the size range of exosomes. (c) EM image of exosomes demonstrates cup-shape morphology, size bar=200 nm. CL: cell lysate; E: exosome lysate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507751&req=5

Figure 0001: The NSCLC SK-MES-1 cell line produces exosomes that can be isolated with ultrafiltration of CCM. (a) 5 µg of protein was used for Western blot analysis of isolated exosomes. The presence of canonical exosome proteins, and the absence of Calnexin demonstrates a pure exosome preparation. (b) TRPS analysis demonstrates a size distribution of particles consistent with the size range of exosomes. (c) EM image of exosomes demonstrates cup-shape morphology, size bar=200 nm. CL: cell lysate; E: exosome lysate.
Mentions: The identification of particles as exosomes relies on various criteria. Exosomes range in size from 50 to 100 nm (8), they display a cup-shaped morphology by EM, and proteomic analysis reveals the presence of common proteins (9–11). Figure 1a demonstrates the presence of canonical exosome proteins and the absence of the endoplasmic reticulum protein Calnexin from exosomes isolated using the ultrafiltration protocol (Supplementary Fig. 1). Furthermore, isolated particles exhibit expected morphology and size profiles consistent with pure exosome preparations (Fig. 1b and c).

Bottom Line: Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield.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, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity.

View Article: PubMed Central - PubMed

Affiliation: Tumour Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, Herston, QLD, Australia.

ABSTRACT
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, we 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. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our 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. We 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, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our 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. We 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. We believe this method will have extensive application in the growing field of extracellular vesicle research.

No MeSH data available.


Related in: MedlinePlus