Isolation of exosomes from blood plasma: Qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods

Tamás Baranyai, Kata Herczeg, Zsófia Onódi, István Voszka, K. Módos, Nikolett Marton, György Nagy, Imre Mäger, Matthew J. Wood, Samir El Andaloussi, Zoltán Pálinkás, Vikas Kumar, Péter Nagy, A. Kittel, E. Búzás, P. Ferdinándy, Z. Giricz

Research output: Contribution to journalArticle

150 Citations (Scopus)

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.

Original languageEnglish
Article numbere0145686
JournalPLoS One
Volume10
Issue number12
DOIs
Publication statusPublished - Dec 1 2015

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exosomes
Exosomes
ultracentrifugation
Size exclusion chromatography
Ultracentrifugation
blood plasma
Gel Chromatography
Albumins
Blood
albumins
Plasmas
purity
methodology
Rats
agarose
Impurities
Dynamic light scattering
gel chromatography
Sedimentation
Sepharose

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Isolation of exosomes from blood plasma : Qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods. / Baranyai, Tamás; Herczeg, Kata; Onódi, Zsófia; Voszka, István; Módos, K.; Marton, Nikolett; Nagy, György; Mäger, Imre; Wood, Matthew J.; El Andaloussi, Samir; Pálinkás, Zoltán; Kumar, Vikas; Nagy, Péter; Kittel, A.; Búzás, E.; Ferdinándy, P.; Giricz, Z.

In: PLoS One, Vol. 10, No. 12, e0145686, 01.12.2015.

Research output: Contribution to journalArticle

Baranyai, T, Herczeg, K, Onódi, Z, Voszka, I, Módos, K, Marton, N, Nagy, G, Mäger, I, Wood, MJ, El Andaloussi, S, Pálinkás, Z, Kumar, V, Nagy, P, Kittel, A, Búzás, E, Ferdinándy, P & Giricz, Z 2015, 'Isolation of exosomes from blood plasma: Qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods', PLoS One, vol. 10, no. 12, e0145686. https://doi.org/10.1371/journal.pone.0145686
Baranyai, Tamás ; Herczeg, Kata ; Onódi, Zsófia ; Voszka, István ; Módos, K. ; Marton, Nikolett ; Nagy, György ; Mäger, Imre ; Wood, Matthew J. ; El Andaloussi, Samir ; Pálinkás, Zoltán ; Kumar, Vikas ; Nagy, Péter ; Kittel, A. ; Búzás, E. ; Ferdinándy, P. ; Giricz, Z. / Isolation of exosomes from blood plasma : Qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods. In: PLoS One. 2015 ; Vol. 10, No. 12.
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T2 - Qualitative and quantitative comparison of ultracentrifugation and size exclusion chromatography methods

AU - Baranyai, Tamás

AU - Herczeg, Kata

AU - Onódi, Zsófia

AU - Voszka, István

AU - Módos, K.

AU - Marton, Nikolett

AU - Nagy, György

AU - Mäger, Imre

AU - Wood, Matthew J.

AU - El Andaloussi, Samir

AU - Pálinkás, Zoltán

AU - Kumar, Vikas

AU - Nagy, Péter

AU - Kittel, A.

AU - Búzás, E.

AU - Ferdinándy, P.

AU - Giricz, Z.

PY - 2015/12/1

Y1 - 2015/12/1

N2 - 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.

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