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

Isolation of Extracellular Microvesicles from Cell Culture Medium: Comparative Evaluation of Methods 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry Springer Journals

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

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Publisher
Springer Journals
Copyright
Copyright © 2018 by Pleiades Publishing, Ltd.
Subject
Chemistry; Bioorganic Chemistry; Medicinal Chemistry
ISSN
1990-7508
eISSN
1990-7516
DOI
10.1134/S1990750818020117
Publisher site
See Article on Publisher Site

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.

Journal

Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry – Springer Journals

Published: May 30, 2018

References

  • The Nobel Prize in Physiology or Medicine 2013
    Rothman, J.E.; Schekman, R.W.; Südhof, T.C.
  • bioRxiv
    Zabeo, D.; Cvjetkovic, A.; Lasser, C.; Schorb, M.; Lotvall, J.; Hoog, J.L.
  • Curr. Protocols Cell Biol.
    Thery, C.; Amigorena, S.; Raposo, G.; Clayton, A.