Dataset Information

Isolation and characterization of extracellular vesicles from tissues

ABSTRACT: Extracellular vesicles (EVs) are lipid bi-layered membrane structures released by all cells. Most EV studies have been performed using cell lines or body fluids, and studies on tissue-derived EVs are limited. Here we present a protocol to isolate EV subpopulations directly from tissues. This approach includes enzymatic treatment of dissociated tissues followed by differential ultracentrifugation and density separation. The isolated EV subpopulations were characterized by electron microscopy and RNA profiling. Additionally, their protein cargo was determined with mass spectrometry, Western blot, and ExoView™. The protocol requires extra steps compared to EV isolation from cell culture medium. Tissue-EV isolation can be performed in 22 hours, but a simplified version can be completed in 8 hours. Most experiments with the protocol have used human melanoma metastases, but the protocol can be applied to other cancer and non-cancer tissues. The procedure can be adopted by researchers experienced with cell culture and EV isolation.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Melanoma Cell

SUBMITTER: Proteomics Core Facility

LAB HEAD: Cecilia Lässer

PROVIDER: PXD021694 | Pride | 2020-11-25

REPOSITORIES: Pride

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Publications

Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation.

Crescitelli Rossella R Lässer Cecilia C Jang Su Chul SC Cvjetkovic Aleksander A Malmhäll Carina C Karimi Nasibeh N Höög Johanna L JL Johansson Iva I Johansson Iva I Fuchs Johannes J Thorsell Annika A Gho Yong Song YS Olofsson Bagge R R Lötvall Jan J

Journal of extracellular vesicles 20200211 1

The majority of extracellular vesicle (EV) studies conducted to date have been performed on cell lines with little knowledge on how well these represent the characteristics of EVs <i>in vivo</i>. The aim of this study was to establish a method to isolate and categorize subpopulations of EVs isolated directly from tumour tissue. First we established an isolation protocol for subpopulations of EVs from metastatic melanoma tissue, which included enzymatic treatment (collagenase D and DNase). Small ...[more]

PMID: 32128073

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Project description:Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque. Effects of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with macaque, suggesting opportunities for optimization. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

Dataset Information

Isolation and characterization of extracellular vesicles from tissues

Publications

Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation.

Similar Datasets

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