Skip to main content
SpringerLink
Log in

Isolation of Extracellular Microvesicles from Cell Culture Medium: Comparative Evaluation of Methods

  • Published:
Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry Aims and scope Submit manuscript

Abstract

Extracellular vesicles (EV) are secreted by cells of multicellular organisms. EV mediate specific mode of intercellular communication by “horizontal” exchange of substances and information. This phenomenon seems to have an essential biological significance and became a subject of intensive research. Biogenesis, structural and functional EV features are usually studied in vitro. Several methods of EV isolation from cell culture medium are currently used; however, selection of a particular method may have a significant impact on obtained results. The choice of the optimal method is usually determined by the amount of starting biomaterial and the aims of the research. We have performed a comparative analysis of four different methods of EV isolation from cell culture medium: differential ultracentrifugation, ultracentrifugation with 30% sucrose/D2O “cushion,” precipitation with plant proteins and latex-based immunoaffinity capturing. EV isolated from several human glial cell lines by different approaches were compared in terms of the following parameters: size, concentration, EV morphology, contamination by non-vesicular particles, content of exosomal tetraspanins on the EV surface, content of total proteins, total RNA, and several glioma-associated miRNAs. The applied methods included nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), cryo-electron microscopy, flow cytometry and RT-qPCR. Based on the obtained results, we have developed practical recommendations that may help researchers to make the best choice of the EV isolation method.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Rothman, J.E., Schekman, R.W., and Südhof, T.C., The Nobel Prize in Physiology or Medicine 2013, Press Release, 2013.

    Google Scholar 

  2. Colombo, M., Raposo, G., and Thery, C., Annual Review of Cell and Developmental Biology, 2014, vol. 30, pp. 255–289.

    Article  CAS  PubMed  Google Scholar 

  3. Pupyshev, A.B., Tsitologiya, 2015, vol. 57, no. 8, pp. 551–562.

    CAS  Google Scholar 

  4. Tkach, M. and Thery, C., Cell, 2016, vol. 164, pp. 1226–1232.

    Article  CAS  Google Scholar 

  5. Lener, T., Gimona, M., Aigner, L., Borger, V., Buzas, E., Camussi, G., Chaput, N., Chatterjee, D., Court, F.A., Del Portillo, H.A., O’Driscoll, L., Fais, S., Falcon-Perez, J.M., Felderhoff-Mueser, U., Fraile, L., Gho, Y.S., Gorgens, A., Gupta, R. C., Hendrix, A., Hermann, D.M., Hill, A.F., Hochberg, F., Horn, P.A., de Kleijn, D., Kordelas, L., Kramer, B.W., Kramer-Albers, E.M., Laner-Plamberger, S., Laitinen, S., Leonardi, T., Lorenowicz, M.J., Lim, S.K., Lotvall, J., Maguire, C.A., Marcilla, A., Nazarenko, I., Ochiya, T., Patel, T., Pedersen, S., Pocsfalvi, G., Pluchino, S., Quesenberry, P., Reischl, I.G., Rivera, F.J., Sanzenbacher, R., Schallmoser, K., Slaper-Cortenbach, I., Strunk, D., Tonn, T., Vader, P., van Balkom, B.W., Wauben, M., Andaloussi, S.E., Thery, C., Rohde, E., and Giebel, B., J. Extracellular Vesicles, 2015, vol. 4, p. 30087.

    Article  CAS  Google Scholar 

  6. Cocucci, E. and Meldolesi, J., Trends Cell Biology, 2015, vol. 25, pp. 364–372.

    Article  CAS  Google Scholar 

  7. Grigorieva, A.E., Dyrkheeva, N.S., Bryzgunova, O.E., Tamkovich, S.N., Chelobanov, B.P., and Ryabchikova, E.I., Biomed. Khim., 2017, vol. 63, pp. 91–96. doi 10.18097/PBMC20176301091

    Article  Google Scholar 

  8. Willms, E., Johansson, H.J., Mager, I., Lee, Y., Blomberg, K.E., Sadik, M., Alaarg, A., Smith, C.I., Lehtio, J., El Andaloussi, S., Wood, M.J., and Vader, P., Sci. Rep., 2016, vol. 6, p. 22519.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zabeo, D., Cvjetkovic, A., Lasser, C., Schorb, M., Lotvall, J., and Hoog, J.L., bioRxiv, 2016.

    Google Scholar 

  10. Smith, Z.J., Lee, C., Rojalin, T., Carney, R.P., Hazari, S., Knudson, A., Lam, K., Saari, H., Ibanez, E.L., Viitala, T., Laaksonen, T., Yliperttula, M., and Wachsmann-Hogiu, S., J. Extracellular Vesicles, 2015, vol. 4, p. 28533.

    Article  CAS  Google Scholar 

  11. Lotvall, J., Hill, A.F., Hochberg, F., Buzas, w E.I., Di Vizio, D., Gardiner, C., Gho, Y.S., Kurochkin, I.V., Mathivanan, S., Quesenberry, P., Sahoo, S., Tahara, H., Wauben, M.H., Witwer, K.W., and Thery, C., J. Extracellular Vesicles, 2014, vol. 3, p. 26913.

    Article  Google Scholar 

  12. Gardiner, C., Di Vizio, D., Sahoo, S., Thery, C., Witwer, K.W., Wauben, M., and Hill, A.F., J. Extracellular Vesicles, 2016, vol. 5, p. 32945.

    Article  CAS  Google Scholar 

  13. Samsonov, R., Burdakov, V., Shtam, T., Radzhabova, C.Z., Vasilyev, D., Tsyrlina, E., Titov, S., Ivanov, M., Berstein, L., Filatov, M., Kolesnikov, N., Gil-Henn, H., and Malek, A., Tumour Biology, 2016, vol. 37, pp. 12011–12021.

    Article  CAS  PubMed  Google Scholar 

  14. Samsonov, R., Shtam, T., Burdakov, V., Glotov, A., Tsyrlina, E., Berstein, L., Nosov, A., Evtushenko, V., Filatov, M., and Malek, A., The Prostate, 2016, vol. 76, pp. 68–79.

    Article  CAS  PubMed  Google Scholar 

  15. Shtam, T., Samsonov, R., Kamyshinsky, R., Pantina, R., Verlov, N., Vasiliev, A., Konevega, A.L., and Malek, A.V., AIP Conference Proceedings, 2017, vol. 1882, 020066. doi 10.1063/1.5001645

    Article  CAS  Google Scholar 

  16. Volnitskiy, A.V., Semenova, E.V., Shtam, T.A., Kovalev, R.A., and Filatov, M.V., Cell Tissue Biol., 2014, vol. 8, no. 5, pp. 368–373.

    Article  Google Scholar 

  17. Thery, C., Amigorena, S., Raposo, G., and Clayton, A., Curr. Protocols Cell Biol., 2009, Chapter 3, unit 3 22. 101002/0471143030.cbo32s30

    Google Scholar 

  18. Zhang, Z, Wang, C., Li, T., Liu, Z., and Li, L., Oncology Letts., 2014, vol. 8, pp. 1701–1706.

    Article  CAS  Google Scholar 

  19. Shtam, T.A., Burdakov, V.S., Landa, S.B., Naryzhny, S.N., Bairamukov, V.Y., Malek, A.V., Orlov, Y.N., and Filatov, M.V., Cell Tissue Biol., 2017, vol. 11, pp. 172–179.

    Article  Google Scholar 

  20. Zhou, D., Wan, Y., Xie, D., Wang, Y., Wei, J., Yan, Q., Lu, P., Mo, L., Xie, J., Yang, S., and Qi, X., Exper. Molec. Med., 2015, vol. 47, p. 182.

    Article  CAS  Google Scholar 

  21. Wei, J., Qi, X., Zhan, Q., Zhou, D., Yan, Q., Wang, Y., Mo, L., Wan, Y., Xie, D., Xie, J., and Yang, S., Biomed. Pharmacother., 2015, vol. 71, pp. 112–118.

    Article  CAS  PubMed  Google Scholar 

  22. Gabriely, G., Wurdinger, T., Kesari, S., Esau, C.C., Burchard, J., Linsley, P.S., and Krichevsky, A.M., Molecular Cellular Biol., 2008, vol. 28, pp. 5369–5380.

    Article  CAS  Google Scholar 

  23. Chan, J.A., Krichevsky, A.M., and Kosik, K.S., Cancer Res., 2005, vol. 65, pp. 6029–6033.

    Article  CAS  PubMed  Google Scholar 

  24. Zhang, S., Lai, N., Liao, K., Sun, J., and Lin, Y., Folia Neuropathologica, 2015, vol. 53, pp. 236–244.

    Article  PubMed  Google Scholar 

  25. Lai, N.S., Dong, Q.S., Ding, H., Miao, Z.L., and Lin, Y.C., J. Clinical Neurosci., 2014, vol. 21, pp. 755–760.

    Article  CAS  Google Scholar 

  26. Peltier, H.J. and Latham, G.J., RNA, 2008, vol. 14, pp. 844–852.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Sadegh Masoudi, M., Mehrabian, E., and Mirzaei, H., J. Cell. Biochem., 2017, vol. 119, pp. 1285–1290. doi 10.1002/jcb.26300

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Petersburg Nuclear Physics Institute of National Research Centre Kurchatov Institute, Saint-Petersburg, Gatchina, 188300, Russia

    T. A. Shtam, A. V. Volnitskiy, N. A. Verlov & A. L. Konevega

  2. Oncosystem Ltd., Skolkovo, 143026, Russia

    T. A. Shtam, R. B. Samsonov & A. V. Malek

  3. Petrov National Medical Research Center of Oncology, St. Petersburg, 197758, Russia

    T. A. Shtam, R. B. Samsonov & A. V. Malek

  4. National Research Center Kurchatov Institute, Moscow, 123182, Russia

    R. A. Kamyshinsky, A. S. Orehov & A. L. Vasiliev

  5. Peter the Great Saint-Petersburg Polytechnic University, St. Petersburg, 195251, Russia

    T. A. Shtam, M. S. Kniazeva, E. A. Korobkina & A. L. Konevega

Authors
  1. T. A. Shtam
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. B. Samsonov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. V. Volnitskiy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R. A. Kamyshinsky
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. N. A. Verlov
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M. S. Kniazeva
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. E. A. Korobkina
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A. S. Orehov
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. A. L. Vasiliev
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. A. L. Konevega
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. A. V. Malek
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to T. A. Shtam.

Additional information

Original Russian Text © T.A. Shtam, R.B. Samsonov, A.V. Volnitskiy, R.A. Kamyshinsky, N.A. Verlov, M.S. Kniazeva, E.A. Korobkina, A.S. Orehov, A.L. Vasiliev, A.L. Konevega, A.V. Malek, 2018, published in Biomeditsinskaya Khimiya.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shtam, T.A., Samsonov, R.B., Volnitskiy, A.V. et al. Isolation of Extracellular Microvesicles from Cell Culture Medium: Comparative Evaluation of Methods. Biochem. Moscow Suppl. Ser. B 12, 167–175 (2018). https://doi.org/10.1134/S1990750818020117

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S1990750818020117

Keywords

Search

Navigation