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PLoS One. 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.

Author information

1
Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary.
2
Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary.
3
Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary.
4
Department of Rheumatology, Polyclinic of the Hospitaller Brothers of St John of God, Budapest, Hungary.
5
Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.
6
Institute of Technology, University of Tartu, Tartu, Estonia.
7
Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
8
Department of Molecular Immunology and Toxicology, National Institute of Oncology, Budapest, Hungary.
9
Centre for Cellular and Molecular Platforms, NCBS, Bangalore, India.
10
Department of Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary.
11
Pharmahungary Group, Budapest, Hungary.

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.

PMID:
26690353
PMCID:
PMC4686892
DOI:
10.1371/journal.pone.0145686
[Indexed for MEDLINE]
Free PMC Article

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