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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 of CCM results in higher recovery of particles after density gradient purification. (a) Particle analysis of 1 mL fractions collected from density gradient. Ultracentrifugation results in a significantly higher proportion of particles at higher densities in fractions 8–10. (b) Total particles (<100 nm) isolated from ultracentrifugation and ultrafiltration. Ultrafiltration of CCM before density gradient purification results in a higher yield of <100 nm particles compared to ultracentrifugation preparation. (c) Percentage recovery of particles collected from fraction 6 and 7 is higher with the ultrafiltration protocol compared to ultracentrifugation. (d) Size distribution of particles isolated from both protocols indicates no difference in size profile of particles isolated. (e) EM images of exosomes isolated with ultracentrifugation and ultrafiltration protocols, size bar=200 nm. n=3±SEM, *p<0.05. UC: ultracentrifugation; UF: ultrafiltration.
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Figure 0004: Ultrafiltration of CCM results in higher recovery of particles after density gradient purification. (a) Particle analysis of 1 mL fractions collected from density gradient. Ultracentrifugation results in a significantly higher proportion of particles at higher densities in fractions 8–10. (b) Total particles (<100 nm) isolated from ultracentrifugation and ultrafiltration. Ultrafiltration of CCM before density gradient purification results in a higher yield of <100 nm particles compared to ultracentrifugation preparation. (c) Percentage recovery of particles collected from fraction 6 and 7 is higher with the ultrafiltration protocol compared to ultracentrifugation. (d) Size distribution of particles isolated from both protocols indicates no difference in size profile of particles isolated. (e) EM images of exosomes isolated with ultracentrifugation and ultrafiltration protocols, size bar=200 nm. n=3±SEM, *p<0.05. UC: ultracentrifugation; UF: ultrafiltration.

Mentions: Next, we examined if repeated ultracentrifugation rounds would reduce the quality and recovery of exosomes purified with a density gradient. Interestingly, ultracentrifugation consistently resulted in significantly higher particle concentrations in fractions 8–10 (p<0.05) compared to exosomes prepared by ultrafiltration of CCM, suggesting that the different preparative methods altered particle characteristics (Fig. 4a). This increase in particle concentration at higher densities for ultracentrifugation was accompanied with an increase in the membrane protein Flotillin-1, but not TSG101, potentially indicating ruptured membranes (Supplementary Fig. 4). Positive protein markers for exosomes overlap in fractions 6 and 7, and these fractions were pooled and ultracentrifuged at 100,000 g for 2 hours to pellet exosomes for further analysis. Both ultracentrifugation and ultrafiltration before OptiPrep™ did not alter the size distribution, or morphology of purified exosomes (Fig. 4d and e). However, ultrafiltration preparations would consistently produce significantly higher particle yields (Fig. 4b), a result that was accompanied by a higher percentage recovery when total particles from fraction 6 and 7 were compared to total particles recovered (Fig. 4c).


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 of CCM results in higher recovery of particles after density gradient purification. (a) Particle analysis of 1 mL fractions collected from density gradient. Ultracentrifugation results in a significantly higher proportion of particles at higher densities in fractions 8–10. (b) Total particles (<100 nm) isolated from ultracentrifugation and ultrafiltration. Ultrafiltration of CCM before density gradient purification results in a higher yield of <100 nm particles compared to ultracentrifugation preparation. (c) Percentage recovery of particles collected from fraction 6 and 7 is higher with the ultrafiltration protocol compared to ultracentrifugation. (d) Size distribution of particles isolated from both protocols indicates no difference in size profile of particles isolated. (e) EM images of exosomes isolated with ultracentrifugation and ultrafiltration protocols, size bar=200 nm. n=3±SEM, *p<0.05. UC: ultracentrifugation; UF: ultrafiltration.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0004: Ultrafiltration of CCM results in higher recovery of particles after density gradient purification. (a) Particle analysis of 1 mL fractions collected from density gradient. Ultracentrifugation results in a significantly higher proportion of particles at higher densities in fractions 8–10. (b) Total particles (<100 nm) isolated from ultracentrifugation and ultrafiltration. Ultrafiltration of CCM before density gradient purification results in a higher yield of <100 nm particles compared to ultracentrifugation preparation. (c) Percentage recovery of particles collected from fraction 6 and 7 is higher with the ultrafiltration protocol compared to ultracentrifugation. (d) Size distribution of particles isolated from both protocols indicates no difference in size profile of particles isolated. (e) EM images of exosomes isolated with ultracentrifugation and ultrafiltration protocols, size bar=200 nm. n=3±SEM, *p<0.05. UC: ultracentrifugation; UF: ultrafiltration.
Mentions: Next, we examined if repeated ultracentrifugation rounds would reduce the quality and recovery of exosomes purified with a density gradient. Interestingly, ultracentrifugation consistently resulted in significantly higher particle concentrations in fractions 8–10 (p<0.05) compared to exosomes prepared by ultrafiltration of CCM, suggesting that the different preparative methods altered particle characteristics (Fig. 4a). This increase in particle concentration at higher densities for ultracentrifugation was accompanied with an increase in the membrane protein Flotillin-1, but not TSG101, potentially indicating ruptured membranes (Supplementary Fig. 4). Positive protein markers for exosomes overlap in fractions 6 and 7, and these fractions were pooled and ultracentrifuged at 100,000 g for 2 hours to pellet exosomes for further analysis. Both ultracentrifugation and ultrafiltration before OptiPrep™ did not alter the size distribution, or morphology of purified exosomes (Fig. 4d and e). However, ultrafiltration preparations would consistently produce significantly higher particle yields (Fig. 4b), a result that was accompanied by a higher percentage recovery when total particles from fraction 6 and 7 were compared to total particles recovered (Fig. 4c).

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