Characterizing extracellular vesicles from cerebrospinal fluid by a novel size exclusion chromatography method

Abstract

AbstractBackgroundExtracellular vesicles (EVs) have recently been considered as a potential biomarker source for a variety of diseases, including neurodegenerative disorders. EVs are important mediators of intercellular communication due to their capacity to transfer genetic material, lipids and proteins. By means of their communication role, interesting biomarkers are often enriched in EVs compared to total biofluid. In order to isolate EVs from a biofluid, like cerebrospinal fluid (CSF), a variety of isolation methods are available of which each methods shows different results, and, to date, there is no golden standard. Our study evaluates the use of size exclusion chromatography (SEC) by the SmartSEC HT kit for EV isolation from CSF.MethodEVs were obtained from 500µL CSF samples, isolated by SmartSEC HT. SmartSEC traps contaminants, like bulk proteins, into the resin beads and subsequently elutes EVs. To evaluate this isolation method, various characterisation analyses were run. To detect cargo or surface proteïns, analyses on the ExoView R100, Surface Plasmon Resonance imaging (SPRi) and LC‐MS based proteomics analysis were performed. Concentration, and size of the vesicles were evaluated by use of transmission electron microscopy (TEM), and fluorescence nanotracking analysis (Zetaview).ResultBy means of the ExoView, tetraspanins such as CD81 and CD9 were measured on the surface of the EVs. Other EV‐specific proteins, e.g. lactadherin, flotillin‐1, and HSP70, were detected by SPRI. Untargeted protein analysis revealed the cargo proteins. TEM images visualized the presence of EVs. Particle size and concentration was calculated by nanotracking analysis, and more specifically the particles with a lipid membrane, like EVs, with fluorescence nanotracking analysis.ConclusionAs EVs hold a lot of promise in the biomarker field, a standard high‐performance isolation method ‐ regarding purity and yield – is essential. From our data, SmartSEC HT is suggested to be an efficient size exclusion chromatography EV isolation method for low volume CSF samples, especially for high throughput, since samples are loaded on a 96‐well plate.