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Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues
Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvi...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432511/ https://www.ncbi.nlm.nih.gov/pubmed/32759826 http://dx.doi.org/10.3390/ijms21155586 |
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author | Bordas, Marie Genard, Géraldine Ohl, Sibylle Nessling, Michelle Richter, Karsten Roider, Tobias Dietrich, Sascha Maaß, Kendra K. Seiffert, Martina |
author_facet | Bordas, Marie Genard, Géraldine Ohl, Sibylle Nessling, Michelle Richter, Karsten Roider, Tobias Dietrich, Sascha Maaß, Kendra K. Seiffert, Martina |
author_sort | Bordas, Marie |
collection | PubMed |
description | Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches—(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols. |
format | Online Article Text |
id | pubmed-7432511 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-74325112020-08-24 Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues Bordas, Marie Genard, Géraldine Ohl, Sibylle Nessling, Michelle Richter, Karsten Roider, Tobias Dietrich, Sascha Maaß, Kendra K. Seiffert, Martina Int J Mol Sci Article Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches—(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols. MDPI 2020-08-04 /pmc/articles/PMC7432511/ /pubmed/32759826 http://dx.doi.org/10.3390/ijms21155586 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Bordas, Marie Genard, Géraldine Ohl, Sibylle Nessling, Michelle Richter, Karsten Roider, Tobias Dietrich, Sascha Maaß, Kendra K. Seiffert, Martina Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues |
title | Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues |
title_full | Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues |
title_fullStr | Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues |
title_full_unstemmed | Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues |
title_short | Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues |
title_sort | optimized protocol for isolation of small extracellular vesicles from human and murine lymphoid tissues |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432511/ https://www.ncbi.nlm.nih.gov/pubmed/32759826 http://dx.doi.org/10.3390/ijms21155586 |
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