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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 choice of concentrating method impacts on particle recovery. (a) Significant reduction in the yield of <100 nm particles was observed with the first run of a membrane using the pressure-driven concentrating Stirred Cell device, but not the centrifuge-based Centricon device. (b) Particle yield from membranes restored with NaOH remained low with the Stirred Cell, but was not altered with the Centricon concentrator. (c) <100 nm particle yield from the Stirred Cell was comparable to Centricon concentration when membranes were washed with ethanol. n=3±SEM, ***p<0.001. SC: Stirred Cell.
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Figure 0002: The choice of concentrating method impacts on particle recovery. (a) Significant reduction in the yield of <100 nm particles was observed with the first run of a membrane using the pressure-driven concentrating Stirred Cell device, but not the centrifuge-based Centricon device. (b) Particle yield from membranes restored with NaOH remained low with the Stirred Cell, but was not altered with the Centricon concentrator. (c) <100 nm particle yield from the Stirred Cell was comparable to Centricon concentration when membranes were washed with ethanol. n=3±SEM, ***p<0.001. SC: Stirred Cell.

Mentions: Currently, the main protein concentrating devices available are either pressure-driven (Stirred Cell) or centrifugation-based (Centricon). In order to investigate if there are differences in exosome yield between the 2 methods, we compared the Stirred Cell using a cellulose or biomax membrane with Centricon protein concentrators. Interestingly, using the Stirred Cell, both cellulose and biomax membranes recovered less particles than the Centricon device, which had an approximate 3-fold greater yield (Fig. 2a). This was also seen when the abundance of the canonical exosome protein Flotillin-1 (9,10) was assessed (Fig. 2b). When membranes were restored with sodium hydroxide, particle yield remained low in the Stirred Cell concentration workflow, with no impact on the yield from the Centricon (Fig. 2b). However, when the membranes were washed thoroughly with ethanol and PBS, the yield from the Stirred Cell increased to levels comparable to the Centricon (Fig. 2c). These data indicated that the Centricon method is superior in concentrating exosomes and that Stirred Cell membranes non-specifically bind a significant amount of exosomes.


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 choice of concentrating method impacts on particle recovery. (a) Significant reduction in the yield of <100 nm particles was observed with the first run of a membrane using the pressure-driven concentrating Stirred Cell device, but not the centrifuge-based Centricon device. (b) Particle yield from membranes restored with NaOH remained low with the Stirred Cell, but was not altered with the Centricon concentrator. (c) <100 nm particle yield from the Stirred Cell was comparable to Centricon concentration when membranes were washed with ethanol. n=3±SEM, ***p<0.001. SC: Stirred Cell.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0002: The choice of concentrating method impacts on particle recovery. (a) Significant reduction in the yield of <100 nm particles was observed with the first run of a membrane using the pressure-driven concentrating Stirred Cell device, but not the centrifuge-based Centricon device. (b) Particle yield from membranes restored with NaOH remained low with the Stirred Cell, but was not altered with the Centricon concentrator. (c) <100 nm particle yield from the Stirred Cell was comparable to Centricon concentration when membranes were washed with ethanol. n=3±SEM, ***p<0.001. SC: Stirred Cell.
Mentions: Currently, the main protein concentrating devices available are either pressure-driven (Stirred Cell) or centrifugation-based (Centricon). In order to investigate if there are differences in exosome yield between the 2 methods, we compared the Stirred Cell using a cellulose or biomax membrane with Centricon protein concentrators. Interestingly, using the Stirred Cell, both cellulose and biomax membranes recovered less particles than the Centricon device, which had an approximate 3-fold greater yield (Fig. 2a). This was also seen when the abundance of the canonical exosome protein Flotillin-1 (9,10) was assessed (Fig. 2b). When membranes were restored with sodium hydroxide, particle yield remained low in the Stirred Cell concentration workflow, with no impact on the yield from the Centricon (Fig. 2b). However, when the membranes were washed thoroughly with ethanol and PBS, the yield from the Stirred Cell increased to levels comparable to the Centricon (Fig. 2c). These data indicated that the Centricon method is superior in concentrating exosomes and that Stirred Cell membranes non-specifically bind a significant amount of exosomes.

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