Background Extracellular vesicles (EVs) play an essential role in the communication between cells and transport of diagnostically significant molecules. A wide diversity of approaches utilizing different biochemical properties of EVs and a lack of accepted protocols make data interpretation very challenging. Scope of Review This review consolidates the data on the classical and state-of-the-art methods for isolation of EVs, including exosomes, highlighting the advantages and disadvantages of each method. Various characteristics of individual methods, including isolation efficiency, EV yield, properties of isolated EVs, and labor consumption are compared. Major Conclusions A mixed population of vesicles is obtained in most studies of EVs for all used isolation methods. The properties of an analyzed sample should be taken into account when planning an experiment aimed at studying and using these vesicles. The problem of adequate EVs isolation methods still remains; it might not be possible to develop a universal EV isolation method but the available protocols can be used towards solving particular types of problems. General Significance With the wide use of EVs for diagnosis and therapy of various diseases the evaluation of existing methods for EV isolation is one of the key problems in modern biology and medicine.

Recent studies suggest that extracellular vesicles may be the key to timely diagnosis and monitoring of genito-urological malignancies. In this study we investigated the composition and content of extracellular vesicles found in the urine of healthy donors and prostate cancer patients. Urine of 14 PCa patients and 20 healthy volunteers was clarified by low-speed centrifugation and total extracellular vesicles fraction was obtain by high-speed centrifugation. The exosome-enriched fraction was obtained by filtration of total extracellular vesicles through a 0.1 μm pore filter. Transmission electron microscopy showed that cell-free urine in both groups contained vesicles from 20 to 230 nm. Immunogold staining after ultrafiltration demonstrated that 95% and 90% of extracellular vesicles in healthy individuals and cancer patients, respectively, were exosomes. Protein, DNA and RNA concentrations as well as size distribution of extracellular vesicles in both fractions were analyzed. Only 75% of the total protein content of extracellular vesicles was associated with exosomes which amounted to 90–95% of all vesicles. Median DNA concentrations in total extracellular vesicles and exosome-enriched fractions were 18 pg/ml and 2.6 pg/ml urine, correspondingly. Urine extracellular vesicles carried a population of RNA molecules 25 nt to 200 nt in concentration of no more than 290 pg/ml of urine. Additionally, concentrations of miR-19b, miR-25, miR-125b, and miR-205 were quantified by qRT-PCR. MiRNAs were shown to be differently distributed between different fractions of extracellular vesicles. Detection of miR-19b versus miR-16 in total vesicles and exosome-enriched fractions achieved 100%/93% and 95%/79% specificity/sensitivity in distinguishing cancer patients from healthy individuals, respectively, demonstrating the diagnostic value of urine extracellular vesicles.

The concentration of circulating DNA (cirDNA) and deoxyribonuclease activity in blood plasma of healthy donors and patients with colon or stomach cancer were analyzed. The concentration of DNA was measured using Hoechst 33258 fluorescent assay after the isolation by the glass-milk protocol. A 1-kbp PCR product labeled with biotinylated forward and fluorescein-labeled reverse primers was used as a substrate for DNase. DNase activity was estimated from the data of immunochemical detection of the nonhydrolyzed amplicon. The average concentration of cirDNA in the plasma of healthy donors was low (34 +/- 34 ng/mL), and was accompanied with high DNase activity (0.356 +/- 0.410 U/mL). The increased concentrations of cirDNA in blood plasma of patients with colon and stomach cancer were accompanied by a decrease in DNase activity below the detection level of the assay. The data obtained demonstrate that low DNase activity in blood plasma of cancer patients can cause an increase in the concentration of cirDNA.