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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

Ultrafiltration recovers more particles compared to ultracentrifugation. (a) Size distribution of particles before density gradient purification. (b) Percentage of particle size ranges from ultracentrifugation and ultrafiltration isolations. (c) Ultrafiltration was shown to significantly increase the recovery of <100 nm particles compared to ultracentrifugation; n=3±SEM, *p<0.05. (d) Western blot analysis of equal volumes from ultracentrifugation and ultrafiltration did not show a large difference in protein markers for exosomes. UC: ultracentrifugation; UF: ultrafiltration.
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Figure 0003: Ultrafiltration recovers more particles compared to ultracentrifugation. (a) Size distribution of particles before density gradient purification. (b) Percentage of particle size ranges from ultracentrifugation and ultrafiltration isolations. (c) Ultrafiltration was shown to significantly increase the recovery of <100 nm particles compared to ultracentrifugation; n=3±SEM, *p<0.05. (d) Western blot analysis of equal volumes from ultracentrifugation and ultrafiltration did not show a large difference in protein markers for exosomes. UC: ultracentrifugation; UF: ultrafiltration.

Mentions: To investigate the exact differences between ultracentrifugation and ultrafiltration on exosome yield and quality, we used a combination of particle analysis and protein assessment of positive markers. Particle tracking using TRPS showed that both ultracentrifugation and ultrafiltration isolated particles ranging in size from 50 to 250 nm (Fig. 3a), with no difference observed in the percentage makeup of populations with defined size ranges (Fig. 3b). Interestingly, ultrafiltration resulted in the highest recovery of particles <100 nm compared to ultracentrifugation (Fig. 3c). Analysis of equal volumes of exosome preparations for HSP70 and Flotillin-1 was not sensitive enough to detect a higher ratio of exosomes between the 2 preparation methods; however, TSG101 expression was increased slightly in the ultrafiltration sample (Fig. 3d). Both methods were therefore comparable in recovering exosomes, yet ultrafiltration was far more time efficient taking only 20 minutes to concentrate 150 mL of CCM compared to 2 rounds of ultracentrifugation for 90 minutes each.


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)

Ultrafiltration recovers more particles compared to ultracentrifugation. (a) Size distribution of particles before density gradient purification. (b) Percentage of particle size ranges from ultracentrifugation and ultrafiltration isolations. (c) Ultrafiltration was shown to significantly increase the recovery of <100 nm particles compared to ultracentrifugation; n=3±SEM, *p<0.05. (d) Western blot analysis of equal volumes from ultracentrifugation and ultrafiltration did not show a large difference in protein markers for exosomes. UC: ultracentrifugation; UF: ultrafiltration.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0003: Ultrafiltration recovers more particles compared to ultracentrifugation. (a) Size distribution of particles before density gradient purification. (b) Percentage of particle size ranges from ultracentrifugation and ultrafiltration isolations. (c) Ultrafiltration was shown to significantly increase the recovery of <100 nm particles compared to ultracentrifugation; n=3±SEM, *p<0.05. (d) Western blot analysis of equal volumes from ultracentrifugation and ultrafiltration did not show a large difference in protein markers for exosomes. UC: ultracentrifugation; UF: ultrafiltration.
Mentions: To investigate the exact differences between ultracentrifugation and ultrafiltration on exosome yield and quality, we used a combination of particle analysis and protein assessment of positive markers. Particle tracking using TRPS showed that both ultracentrifugation and ultrafiltration isolated particles ranging in size from 50 to 250 nm (Fig. 3a), with no difference observed in the percentage makeup of populations with defined size ranges (Fig. 3b). Interestingly, ultrafiltration resulted in the highest recovery of particles <100 nm compared to ultracentrifugation (Fig. 3c). Analysis of equal volumes of exosome preparations for HSP70 and Flotillin-1 was not sensitive enough to detect a higher ratio of exosomes between the 2 preparation methods; however, TSG101 expression was increased slightly in the ultrafiltration sample (Fig. 3d). Both methods were therefore comparable in recovering exosomes, yet ultrafiltration was far more time efficient taking only 20 minutes to concentrate 150 mL of CCM compared to 2 rounds of ultracentrifugation for 90 minutes each.

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