Limits...
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

Alternative rapid isolation techniques of exosomes from concentrated media. (a) Size distribution and EM images of particles isolated from precipitation methods and qEV SEC columns, size bar=200 nm. (b) Precipitation methods isolate significantly more particles (<100 nm) compared to SEC and density gradient purification. (c) Concentration of particles expressed as a ratio per microgram of protein. Both SEC and DG provide superior purity as illustrated by significantly more particles per microgram of protein compared to precipitation protocols. (d) No difference was observed in the particle size composition of different isolation methods. (e) Western blot analysis of 10 µg of protein from each protocol. Exosome-positive markers were enriched in qEV and DG lysates compared to precipitation isolations, and all isolation techniques were absent for Calnexin, which was present only in the cell lystate fraction. n=3±SEM, *p<0.05, **p<0.01, ***p<0.001. CL: cell lysate; EQ: ExoQuick™; ES: Exo-spin™; qEV: size exclusion columns; DG: density gradient.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4507751&req=5

Figure 0005: Alternative rapid isolation techniques of exosomes from concentrated media. (a) Size distribution and EM images of particles isolated from precipitation methods and qEV SEC columns, size bar=200 nm. (b) Precipitation methods isolate significantly more particles (<100 nm) compared to SEC and density gradient purification. (c) Concentration of particles expressed as a ratio per microgram of protein. Both SEC and DG provide superior purity as illustrated by significantly more particles per microgram of protein compared to precipitation protocols. (d) No difference was observed in the particle size composition of different isolation methods. (e) Western blot analysis of 10 µg of protein from each protocol. Exosome-positive markers were enriched in qEV and DG lysates compared to precipitation isolations, and all isolation techniques were absent for Calnexin, which was present only in the cell lystate fraction. n=3±SEM, *p<0.05, **p<0.01, ***p<0.001. CL: cell lysate; EQ: ExoQuick™; ES: Exo-spin™; qEV: size exclusion columns; DG: density gradient.

Mentions: Exosomes were prepared from concentrated CCM of SK-MES-1 cells using 4 different isolation techniques. With the recent increased interest in EV research, there have been numerous commercial products being developed for the rapid isolation of exosomes. We compared 3 commercially available products: ExoQuick™, Exo-spin™ and Izon qEV columns with OptiPrep™ density gradient prepared exosomes. All 3 commercial products provided very similar size distribution profiles and morphology (Fig. 5a), the only difference being the presence of particles >200 nm when compared to density gradient exosome preparations (Fig. 5a and d). ExoQuick™ and Exo-spin™ produced significantly higher yields of <100 nm particles compared to qEV and density gradient isolation techniques (Fig. 5b). By contrast, when particles are expressed per µg of protein (a good indicator of particle purity (12)), ExoQuick™ is shown to perform poorly, suggesting the co-isolation of contaminating proteins (Fig. 5c). Exo-spin™ performed significantly better compared to ExoQuick™, but both qEV and density gradient are the superior isolation techniques (Fig. 5c). Although there is a potential that precipitation protocols co-isolate a higher percentage of larger particles, we do not find this (Fig. 5d), largely due to the preparation of CCM with a 0.22 µm filtration step. The absence of larger particles is further validated with a larger nanopore (Supplementary Fig. 5). The increased ratio of particles per µg of protein seen with qEV columns compared to density gradient purification is most likely due to the loss of particles associated with density gradient purification (Fig. 4a). When selecting purely on size this loss is avoided, providing higher recovery of particles <100 nm in size.


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)

Alternative rapid isolation techniques of exosomes from concentrated media. (a) Size distribution and EM images of particles isolated from precipitation methods and qEV SEC columns, size bar=200 nm. (b) Precipitation methods isolate significantly more particles (<100 nm) compared to SEC and density gradient purification. (c) Concentration of particles expressed as a ratio per microgram of protein. Both SEC and DG provide superior purity as illustrated by significantly more particles per microgram of protein compared to precipitation protocols. (d) No difference was observed in the particle size composition of different isolation methods. (e) Western blot analysis of 10 µg of protein from each protocol. Exosome-positive markers were enriched in qEV and DG lysates compared to precipitation isolations, and all isolation techniques were absent for Calnexin, which was present only in the cell lystate fraction. n=3±SEM, *p<0.05, **p<0.01, ***p<0.001. CL: cell lysate; EQ: ExoQuick™; ES: Exo-spin™; qEV: size exclusion columns; DG: density gradient.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0005: Alternative rapid isolation techniques of exosomes from concentrated media. (a) Size distribution and EM images of particles isolated from precipitation methods and qEV SEC columns, size bar=200 nm. (b) Precipitation methods isolate significantly more particles (<100 nm) compared to SEC and density gradient purification. (c) Concentration of particles expressed as a ratio per microgram of protein. Both SEC and DG provide superior purity as illustrated by significantly more particles per microgram of protein compared to precipitation protocols. (d) No difference was observed in the particle size composition of different isolation methods. (e) Western blot analysis of 10 µg of protein from each protocol. Exosome-positive markers were enriched in qEV and DG lysates compared to precipitation isolations, and all isolation techniques were absent for Calnexin, which was present only in the cell lystate fraction. n=3±SEM, *p<0.05, **p<0.01, ***p<0.001. CL: cell lysate; EQ: ExoQuick™; ES: Exo-spin™; qEV: size exclusion columns; DG: density gradient.
Mentions: Exosomes were prepared from concentrated CCM of SK-MES-1 cells using 4 different isolation techniques. With the recent increased interest in EV research, there have been numerous commercial products being developed for the rapid isolation of exosomes. We compared 3 commercially available products: ExoQuick™, Exo-spin™ and Izon qEV columns with OptiPrep™ density gradient prepared exosomes. All 3 commercial products provided very similar size distribution profiles and morphology (Fig. 5a), the only difference being the presence of particles >200 nm when compared to density gradient exosome preparations (Fig. 5a and d). ExoQuick™ and Exo-spin™ produced significantly higher yields of <100 nm particles compared to qEV and density gradient isolation techniques (Fig. 5b). By contrast, when particles are expressed per µg of protein (a good indicator of particle purity (12)), ExoQuick™ is shown to perform poorly, suggesting the co-isolation of contaminating proteins (Fig. 5c). Exo-spin™ performed significantly better compared to ExoQuick™, but both qEV and density gradient are the superior isolation techniques (Fig. 5c). Although there is a potential that precipitation protocols co-isolate a higher percentage of larger particles, we do not find this (Fig. 5d), largely due to the preparation of CCM with a 0.22 µm filtration step. The absence of larger particles is further validated with a larger nanopore (Supplementary Fig. 5). The increased ratio of particles per µg of protein seen with qEV columns compared to density gradient purification is most likely due to the loss of particles associated with density gradient purification (Fig. 4a). When selecting purely on size this loss is avoided, providing higher recovery of particles <100 nm in size.

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