1. Petersburg Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, Saint-Petersburg, Gatchina, Russia; Oncosystem” Ltd., Skolkovo, Russia; N.N.Petrov National Medical Research center of Oncology, Saint-Petersburg, Russia; Peter the Great Saint-Petersburg Polytechnic University, St. Petersburg, Russia 2. Oncosystem” Ltd., Skolkovo, Russia; N.N.Petrov National Medical Research center of Oncology, Saint-Petersburg, Russia 3. Petersburg Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, Saint-Petersburg, Gatchina, Russia 4. National Research Center “Kurchatov Institute”, Moscow, Russia 5. Peter the Great Saint-Petersburg Polytechnic University, St. Petersburg, Russia 6. Petersburg Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, Saint-Petersburg, Gatchina, Russia; Peter the Great Saint-Petersburg Polytechnic University, St. Petersburg, Russia
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 features of the EV is being commonly studies in in vitro condition. Several methods of EV isolation from cell culture medium are established, however selection of method might influence on obtained results. The choice of the optimal method depends usually from the amount of medium and the aims of the research while is still challenging issue. We performed a comparative analysis of four different methods of EV isolation from cell culture medium: differential ultracentrifugation, ultracentrifugation with a 30% sucrose/D2O “cushion”, precipitation with plant proteins and immune-affinity capturing. EV isolated by different approaches were compared in terms of following parameters: size, concentration, morphology of EV, contamination by non-vesicular particles, content of exosomal tetraspanins on the EV surface, content of total proteins, RNA, and several glioma-associated miRNAs. Applied methods included nano-patricle tracking analysis (NTA), dynamic light scattering (DLS), cryo-electron microscopy, flow cytometry and RT-qPCR. On the base of obtained results, we developed practical recommendations that may help researchers to make a best choice of EV isolation method.
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Keywords: extracellular vesicles, exsosomes, methods of isolation, ultracentrifugation, lectines, immunoprecipitation
Shtam T.A. et al. Isolation of extracellular micro-vesicles from cell culture medium: comparative evaluation of methods // Biomeditsinskaya Khimiya. - 2018. - V. 64. -N 1. - P. 23-30.
Shtam T.A. et al., "Isolation of extracellular micro-vesicles from cell culture medium: comparative evaluation of methods." Biomeditsinskaya Khimiya 64.1 (2018): 23-30.
Shtam, T. A., Samsonov, R. A., Volnitskiy, A. V., Kamyshinsky, R. A., Verlov, N. A., Kniazeva, M. S., Korobkina, E. A., Orehov, A. S., Vasiliev, A. L., Konevega, A. L., Malek, A. V. (2018). Isolation of extracellular micro-vesicles from cell culture medium: comparative evaluation of methods. Biomeditsinskaya Khimiya, 64(1), 23-30.
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