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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

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.
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Figure 0006: 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.

Mentions: In order to assess the utility of these techniques on the isolation of exosomes from human bodily fluids, we isolated exosomes from normal human plasma using ExoQuick™, Exo-spin™ and qEV columns. Density gradient purification has been shown to provide the most pure exosome preparation from plasma (13); however, due to the often-limited sample availability of plasma, we decided to assess techniques that would not require more than 1 mL of plasma. All 3 methods isolate particles with a size range equivalent to exosomes (Fig. 6a), but recovery of particles from plasma is dependent on the isolation process (Fig. 6b). At a low purity level, both precipitation protocols recover the majority of particles in relation to unprocessed plasma (Fig. 6b). The SEC method using qEV columns provided the lowest exosome recovery rate (Fig. 6b), but with the highest purity, based on the selective inclusion of eluted fractions. Similar to the isolation of exosomes from CCM, when the concentration of particles is expressed relative to protein concentration, ExoQuick™ performs very poorly (Fig. 6c). Exo-spin™ provides a significantly higher purification compared to ExoQuick™, but column purification alone provides the highest degree of purification (Fig. 6c). This is supported by EM, and Western blot analysis of 50 µg of protein. EM images indicate a high degree of contamination in exosome isolations (Fig. 6a), and Flotillin-1 was only detectable in the samples isolated with qEV technique (Fig. 6d). This was further accompanied with a high degree of albumin contamination in the ExoQuick™ and Exo-spin™ that was not present in the qEV isolation (Fig. 6d).


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0006: 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.
Mentions: In order to assess the utility of these techniques on the isolation of exosomes from human bodily fluids, we isolated exosomes from normal human plasma using ExoQuick™, Exo-spin™ and qEV columns. Density gradient purification has been shown to provide the most pure exosome preparation from plasma (13); however, due to the often-limited sample availability of plasma, we decided to assess techniques that would not require more than 1 mL of plasma. All 3 methods isolate particles with a size range equivalent to exosomes (Fig. 6a), but recovery of particles from plasma is dependent on the isolation process (Fig. 6b). At a low purity level, both precipitation protocols recover the majority of particles in relation to unprocessed plasma (Fig. 6b). The SEC method using qEV columns provided the lowest exosome recovery rate (Fig. 6b), but with the highest purity, based on the selective inclusion of eluted fractions. Similar to the isolation of exosomes from CCM, when the concentration of particles is expressed relative to protein concentration, ExoQuick™ performs very poorly (Fig. 6c). Exo-spin™ provides a significantly higher purification compared to ExoQuick™, but column purification alone provides the highest degree of purification (Fig. 6c). This is supported by EM, and Western blot analysis of 50 µg of protein. EM images indicate a high degree of contamination in exosome isolations (Fig. 6a), and Flotillin-1 was only detectable in the samples isolated with qEV technique (Fig. 6d). This was further accompanied with a high degree of albumin contamination in the ExoQuick™ and Exo-spin™ that was not present in the qEV isolation (Fig. 6d).

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