Tear Fluid Derived Extracellular Vesicle Isolation, Characterization, And Quantification Of Its Protein Cargo

Extracellular vesicles (EVs) carry a diverse range of biomolecules. Tear fluid is a promising source of EVs, and through the minimally invasive nature of tear fluid collection it could provide readily available disease specific information. The objective of this study is to optimize the isolation of extracellular vesicles (EVs) from tear fluid, characterize their biological properties, and quantify their protein cargo.

All study subjects were recruited at the University Eye Clinic Maastricht, Maastricht, the Netherlands. The experimental part was performed at the School of Mental Health and Neuroscience (MHeNs) of the Maastricht University, Maastricht, The Netherlands.

Tear fluid was collected from healthy subjects using Schirmer’s strips. Tear fluid was extracted by elution and centrifugation. EVs present in tear fluid were isolated using ExoQuick at different incubation times (1 hour versus 24 hours) and volumes (0.02, 0.1, 0.5, 1, 2 mL). Particle size distribution and concentration were analysed using Nanoparticle Tracking Analysis (Zetaview^®, Particle Metrix). Protein quantification was performed using the BCA assay. Flow cytometry was used to determine the presence of exosomal markers CD9, CD63 and CD81 on tear fluid derived EVs.

The highest yield of tear fluid EVs (11.17x10⁸ ± 8.52x10⁸ particles/ml) was isolated using 0.5 mL ExoQuick volume. Overnight incubation did not increase the yield. 1.68 ± 0.64% of the total protein content in the tear fluid was loaded into EVs as protein cargo. The isolated EVs were positive for the CD9, CD63 and CD81 demonstrating the presence of exosomes in tear fluid. 

Tear fluid derived EVs and their protein cargos are a promising source of disease specific proteins in ophthalmology.