Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Bordas, Marie, Genard, Géraldine, Ohl, Sibylle, Nessling, Michelle, Richter, Karsten, Roider, Tobias, Dietrich, Sascha, Maaß, Kendra K., Seiffert, Martina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
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
_version_ 1783571815374782464
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
work_keys_str_mv AT bordasmarie optimizedprotocolforisolationofsmallextracellularvesiclesfromhumanandmurinelymphoidtissues
AT genardgeraldine optimizedprotocolforisolationofsmallextracellularvesiclesfromhumanandmurinelymphoidtissues
AT ohlsibylle optimizedprotocolforisolationofsmallextracellularvesiclesfromhumanandmurinelymphoidtissues
AT nesslingmichelle optimizedprotocolforisolationofsmallextracellularvesiclesfromhumanandmurinelymphoidtissues
AT richterkarsten optimizedprotocolforisolationofsmallextracellularvesiclesfromhumanandmurinelymphoidtissues
AT roidertobias optimizedprotocolforisolationofsmallextracellularvesiclesfromhumanandmurinelymphoidtissues
AT dietrichsascha optimizedprotocolforisolationofsmallextracellularvesiclesfromhumanandmurinelymphoidtissues
AT maaßkendrak optimizedprotocolforisolationofsmallextracellularvesiclesfromhumanandmurinelymphoidtissues
AT seiffertmartina optimizedprotocolforisolationofsmallextracellularvesiclesfromhumanandmurinelymphoidtissues