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Comparative Study
. 2015 Dec 21;10(12):e0145686.
doi: 10.1371/journal.pone.0145686. eCollection 2015.

Isolation of Exosomes From Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods

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Free PMC article
Comparative Study

Isolation of Exosomes From Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods

Tamás Baranyai et al. PLoS One. .
Free PMC article

Abstract

Background: Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described.

Aim: Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC).

Methods and results: Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin.

Conclusion: Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Exosomes can be isolated with 1h ultracentrifugation method from blood plasma.
(A) Transmission electron microscopy images of exosome isolates from rat and human blood plasma. (B) CD63, TSG101 and albumin content of the rat and human exosomal isolates as evaluated with Western blot. (C) Size distribution of particles isolated from rat and human blood plasma analyzed with dynamic light scattering (averages of n = 3–4).
Fig 2
Fig 2. Efficiency of ultracentrifugation (UC) methods under various conditions.
(A) Scheme of serial UC of the supernatant. Exosomal marker CD63 and TSG101 and albumin content of exosomal isolates after serial UC of the supernatant with 1h UC at 4°C (B; n = 3; p>0.05), with 10-fold dilution at 4°C (C, top), with smaller volume of loaded sample at 4°C (C, middle) and with 1h UC at 37°C (C, bottom) as evaluated with Western blot.
Fig 3
Fig 3. The effect of various ultracentrifugation (UC) duration on the exosomal yield and purity.
(A) Transmission electron microscopy images of rat exosomes isolated with 1, 3, 6 or 14h UC period. (B) Size distribution of rat exosomes based on transmission electron microscopy image analysis (1h UC: n = 2,440; 3h UC: n = 353). (C) Size distribution of particles isolated with different UC duration evaluated with dynamic light scattering (averages of n = 3). (D) Protein concentration of exosome homogenates isolated with different UC duration as assessed with bicinchoninic acid assay (n = 3–7; *, #, &: p<0.05 vs. other three groups). (E) CD63 and albumin content of exosome isolates with different UC length (n = 3; *, #, &: p<0.05 vs. corresponding other three groups; §: p<0.05 vs. 1h).
Fig 4
Fig 4. Efficiency and selectivity of size exclusion chromatography (SEC) on exosome isolation performed with various matrices.
(A) CD63, TSG101 and albumin content of different fractions collected during SEC on Sepharose 2B (top), Sepharose CL-4B (middle) and Sephacryl S-400 (bottom) columns with equal volumes (left column) or equal protein amounts of fractions (right column) loaded for Western blot. (B) Size distribution of particles isolated with various SEC matrices evaluated with dynamic light scattering. Sepharose 2B (top), Sepharose CL-4B (middle) and Sephacryl S-400 (bottom) columns.
Fig 5
Fig 5. Size exclusion chromatography isolation of exosomes from rat blood plasma on a large-scale Sephacryl S-400 column.
(A) Particle concentration and protein content (absorbance at 280 nm) of eluted fractions. (B) Concentration and size distribution of particles in pooled fractions by nanotracking analysis. (C) CD63, TSG101 and albumin content of pooled

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Grant support

This work was supported by the Hungarian Scientific Research Fund (OTKA PD 109051, OTKA NK 84043, OTKA NK 111958, OTKA K 109843) and the following grants: Marie Curie Networks for Initial Training-ITN-FP7-PEOPLE-2011-ITN, PITN-GA-2011-289033 and COST Action BM1202 ME-HAD. P.N. and Z.G. hold a “János Bolyai Fellowship” from the Hungarian Academy of Sciences. I.M. is supported by the Estonian Research Council Grant PUT618.
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