The aim of our study was to attract the attention of researchers at the problem of contamination of exosome preparations. Using a transmission electron microscope JEM-1400 ("JEOL", Japan) we have examined exosome preparations, isolated according to the conventional scheme of sequential centrifugation from different biological fluids: plasma and urine of healthy persons and patients with oncologic diseases, bovine serum, and culture fluid (MDCK, MDA-MB и MCF-7 cells). All exosome preparations (over 200) contained exosomes, which were identified by immuno-electron microscopy using antibodies to tetraspanins CD63 or CD9. Besides exosomes, all the studied preparations contained contaminating structures: distinct particles of low electron density without limiting membrane ("non-vesicles"). Two main kinds of the "non-vesicles" species were found in exosome preparations: 20-40 nm in size, representing 10-40% of all structures in the preparations; and 40-100 nm in size (identical to exosomes by size). Morphology of the "non-vesicles" allowed to identify them as lipoproteins of intermediate and low density (20-40 nm), and very low density (40-100 nm). The highest level of the contamination was detected in exosome preparations, isolated from blood samples. The results of our study indicate the need to control the composition of exosome preparation by electron microscopy and take into account the presence of contaminating structures in analysis of experimental data.
Tsel'iu nasheĭ raboty bylo privlech' vnimanie issledovateleĭ k probleme kontaminatsii preparatov ékzosom. My izuchali v prosvechivaiushchem élektronnom mikroskope JEM-1400 (“JEOL”, Iaponiia) preparaty ékzosom, vydelennye s pomoshch'iu posledovatel'nykh tsentrifugirovaniĭ po priniatym skhemam iz razlichnykh biologicheskikh zhidkosteĭ: plazmy i mochi zdorovykh i bol'nykh onkologicheskimi zabolevaniiami liudeĭ, syvorotki krupnogo rogatogo skota i kul'tural'noĭ zhidkosti (kletki liniĭ MDCK, MDA-MB i MCF-7). Vse preparaty (bolee 200) soderzhali ékzosomy (vezikuly), identifitsirovannye metodom immunno-élektronnoĭ mikroskopii po sviazyvaniiu s antitelami k tetraspaninam CD63 ili CD9. Krome ékzosom, vo vsekh issledovannykh preparatakh prisutstvovali kontaminiruiushchie struktury nizkoĭ élektronnoĭ plotnosti, ne imeiushchie ogranichivaiushcheĭ membrany i, sootvetstvenno, ne iavliaiushchiesia ékzosomami (“ne-vezikuly”). Otmecheny dva osnovnykh tipa “ne-vezikul”: imeiushchie razmer 20-40 nm i sostavliaiushchie 10-40% vsekh struktur preparatov ékzosom, i imeiushchie razmer 40-100 nm. Morfologiia “ne-vezikul” pozvoliaet otnesti ikh k lipoproteinam promezhutochnoĭ i nizkoĭ plotnosti (20-40 nm) i ochen' nizkoĭ plotnosti (40-100 nm). Poslednie identichny po razmeru ékzosomam. Naibol'shiĭ uroven' kontaminatsii otmechen v preparatakh ékzosom krovi. Poluchennye dannye ukazyvaiut na neobkhodimost' kontrolirovat' sostav preparatov ékzosom s pomoshch'iu élektronnoĭ mikroskopii i uchityvat' prisutstvie kontaminiruiushchikh struktur pri analize i interpretatsii rezul'tatov issledovaniia.
Keywords: electron microscopy; exosomes; lipoproteins.