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

Abstract

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 biolog ical 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 investi gated by electron microscopy and immunolabeling, RNA and DNA were isolated and their sizes were deter mined 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 con tained 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 micro particles (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.

Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis

“…Exosomes, also referred to as intraluminal vesicles (ILVs), are enclosed within a single outer membrane, and are secreted by all cell types and have been found in plasma, urine, semen, saliva, bronchial fluid, cerebral spinal fluid (CSF), breast milk, serum, amniotic fluid, synovial fluid, tears, lymph, bile, and gastric acid [18,22,23,24,29,30,31,32,33,34,35,36,37,38,39].…”

Exosomal Non Coding RNA in LIQUID Biopsies as a Promising Biomarker for Colorectal Cancer

“…Exosomes are phospholipid bilayer nanovesicles, measuring 30-150nm in diameter that contain a range of molecules, including proteins, lipids, and different types of nucleic acid. Various cell types secrete them, including cancerous cells [34,35] and they are present in many biological fluids [36][37][38][39][40][41][42][43][44][45][46][47][48][49]. Endocytic vesicle invagination on the plasma membrane to form an early endosome represents the initiation step of exosomal biogenesis (Figure 1).…”

Signaling of Tumor-Derived sEV Impacts Melanoma Progression

“…sEV are produced and released by various cell types, including hematopoietic cells and a broad variety of normal or malignant tissue cells [ 5 , 7 , 12 , 13 ]. sEV can be isolated from supernatants of cultured cells or diverse body fluids, including blood, urine, saliva, breast milk, ascites effusions, bile, tears, nasal secretions, amniotic, synovial, cerebrospinal, lymphatic, and seminal fluids [ 5 , 7 , 10 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. The molecular content of sEV is of special interest, as it reflects the nature of parental cells.…”