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

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

doi:10.1134/S1990750818020117

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

Authors: Shtam T.A.¹^,2^,3^,4, Samsonov R.B.²^,3, Volnitskiy A.V.¹, Kamyshinsky R.A.⁵, Verlov N.A.¹, Kniazeva M.S.⁴, Korobkina E.A.⁴, Orehov A.S.⁵, Vasiliev A.L.⁵, Konevega A.L.¹^,4, Malek A.V.²^,3
Affiliations:
1. Petersburg Nuclear Physics Institute of National Research Centre Kurchatov Institute
2. Oncosystem Ltd.
3. Petrov National Medical Research Center of Oncology
4. Peter the Great Saint-Petersburg Polytechnic University
5. National Research Center Kurchatov Institute
Issue: Vol 12, No 2 (2018)
Pages: 167-175
Section: Article
URL: https://journals.rcsi.science/1990-7508/article/view/198232
DOI: https://doi.org/10.1134/S1990750818020117
ID: 198232

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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.

Keywords

extracellular vesicles, exsosomes, methods of isolation, ultracentrifugation, lectines, immunoprecipitation

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

Full Text

Abstract

Keywords

About the authors

Supplementary files