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.
Vnekletochnye mikrovezikuly (VMV) sekretiruiutsia kletkami mnogokletochnykh organizmov i obespechivaiut “gorizontal'nyĭ” mezhkletochnyĭ perenos veshchestv i informatsii. Étot fenomen imeet vazhnoe biologicheskoe znachenie i privlekaet vnimanie tysiach issledovateleĭ. Bol'shaia chast' issledovaniĭ provoditsia v usloviiakh in vitro éksperimentov, pozvoliaiushchikh analizirovat' biogenez, strukturnye i funktsional'nye osobennosti VMV. V laboratornoĭ praktike sushchestvuet neskol'ko metodov vydeleniia VMV iz kul'tural'noĭ sredy, prichem vybor metoda vliiaet na poluchaemye rezul'taty. Optimal'nyĭ metod obychno opredeliaetsia ob"emom issleduemogo materiala i zadachami issledovaniia. V ramkakh dannoĭ raboty proveden sravnitel'nyĭ analiz chetyrekh metodov vydeleniia VMV iz kul'tural'noĭ sredy: posledovatel'noe ul'tratsentrifugirovanie, ul'tratsentrifugirovanie s ispol'zovaniem “podushki” sakharozy, aggliutinatsiia VMV lektinami rastitel'nogo proiskhozhdeniia i immunopretsipitatsiia na lateksnykh chastitsakh. V issledovanii byl ispol'zovan riad glial'nykh kletochnykh liniĭ cheloveka. Provedena sravnitel'naia otsenka preparatov VMV, vydelennykh raznymi metodami, po riadu parametrov: razmer, kontsentratsiia, morfologiia VMV, nalichie primeseĭ nevezikuliarnoĭ prirody, soderzhanie v membrane VMV ékzosomal'nykh tetraspaninov, soderzhanie belka, total'noĭ RNK, i neskol'kikh mikroRNK, assotsiirovannykh s razvitiem gliom. Ispol'zovany metody: analiz traektoriĭ nanochastits, lazernaia korreliatsionnaia spektroskopiia, krioélektronnaia mikroskopiia, protochnaia tsitometriia, OT-PTsR. Po rezul'tatam raboty sformulirovany prakticheskie rekomendatsii otnositel'no vybora optimal'nogo metoda s uchetom vozmozhnykh zadach issledovaniia.
Keywords: exsosomes; extracellular vesicles; immunoprecipitation; lectines; methods of isolation; ultracentrifugation.