Contamination of exosome preparations,isolated from biological fluids

The aim of this study was to attract attention of researchers to 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: blood plasma and urine of healthy persons and patients with oncologic diseases, bovine serum, and conditioned cell culture medium (MDCK, MDA-MB, and MCF-7 cells). All examined 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 were characterized by the presence of contaminating structures: low electron density particles without limiting membrane and therefore could not be attributed to exosomes (“non-vesicles”). Two main types of the “non-vesicles” were found in the exosome preparations: particles of 20–40 nm in size, representing 10–40% of all structures in the exosome preparations; and particles of 40–100 nm in size (identical to exosomes by size). Morphology of the “non-vesicles” corresponded to that of intermediate and low density lipoproteins (20–40 nm), and very low density lipoproteins (40–100 nm), which were identical to exosomes in their size. 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 preparations by electron microscopy and to take into consideration the presence of contaminating structures in the analysis of experimental data.