Extraction of Extracellular Vesicles from Whole Tissue

Abstract

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.

Figure 1.. Schematic overview of vesicle isolation and purification from whole tissue.

Following tissue dissociation, pre-clearing differential centrifugation steps, filtration, and ultracentrifugation, crude EV pellets can be resuspended on the bottom of an iodixanol density gradient for floatation separation.

Figure 2.. Representative morphologic and immunophenotypic analysis of tissue-derived EVs.

A) Calculated densities of iodixanol gradient following ultracentrifugation, averaged over independent experiments, highlighting fractions containing light and dense EVs. B) Representative immunoblots of tissue-derived vesicles demonstrating the isolation of predominately dense tumor EVs and light brain EVs. Vesicle isolates are depleted of non-EV protein calnexin. H, tissue homogenate. C) Nanoparticle tracking analysis (NTA) of representative gradient-purified tissue EVs. The smallest detected particle in this sample was 78 nm, and approximately 92% of vesicles detected were 250 nm or smaller, consistent with a high enrichment of small EVs. D) Representative electron microscopy images of brain tissue-derived EVs. Scale bars = 200 nm.

Figure 3.. Summary of highlighted vesicle proteins detected by mass spectrometry of a representative tissue-derived EV sample.

Following EV extraction and gradient purification, 10 μg of EV protein in enriched fraction was loaded into a polyacrylamide gel for electrophoresis and in-gel trypsin digestion. In this representative tissue-derived EV sample, a total of 918 proteins were identified by LC-MS/MS analysis. Peptides were identified, analyzed, and found to be enriched in exosomal, lysosomal, and plasma membrane proteins as previously described. Here we demonstrate the presence of small EV or exosomal proteins in our preparations that have been described by the Théry and Hill labs^,.