Exosomes in tears of healthy individuals: Isolation,identification, and characterization

Exosomes represent a sort of extracellular vesicles, which transfer molecular signals in the body and contain markers of the exosome-producing cell. This study was aimed at search of exosomes in the tears of healthy humans, validation of their nature and examination of their morphological and molecular-biological characteristics. Samples of the tears individually collected from 24 healthy donors (aged 45–60 years) were centrifuged at 20000 g for 15 min to pellet cell debris. The supernatants were examined in an electron microscope using negative staining and were also used for isolation and purification of exosomes by filtration (100 nm pore-size) and double ultracentrifugation (90 min at 100000 g, 4°C). Resultant pellets were investigated by electron microscopy and immunolabeling, RNA and DNA were isolated and their sizes were determined by capillary electrophoresis, concentration and localization of nucleic acids in the isolated exosomes were studied. DNA sequencing was performed using MiSeq (Illumina, USA), data were analyzed using CLC GW 7.5 (Qiagen, USA). Sequences were mapped on human genome (hg19). Supernatants of the tears contained cell debris, spherical microparticles (20–40 nm), and vesicles; some of the vesicles had morphology and sizes corresponding to exosomes. The pellets obtained after ultracentrifugation of tears contained microparticles (17%), spherical and cup-shaped vesicles (40–100 nm, 83%), which were positive for CD63, CD9, and CD24 receptors (specific markers of exosomes). The study revealed high concentrations of exosomes in human tears; these exosomes contained both RNA (of less than 200 nucleotides in size) and DNA (of 3–9 kb in size). DNA sequencing demonstrated that about 92% of the reads was mapped to human genome.