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Open AccessArticle

Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues

1
Division of Molecular Genetics, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
2
Faculty of Biosciences, University of Heidelberg, 69120 Heidelberg, Germany
3
Division of Biomedical Physics in Radiation Oncology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
4
Central Unit Electron Microscopy, DKFZ, 69120 Heidelberg, Germany
5
Department of Medicine V, Hematology, Oncology and Rheumatology, University of Heidelberg, 69120 Heidelberg, Germany
6
Hopp-Children’s Cancer Center Heidelberg (KiTZ), 69120 Heidelberg, Germany
7
Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2020, 21(15), 5586; https://doi.org/10.3390/ijms21155586
Received: 6 July 2020 / Revised: 29 July 2020 / Accepted: 31 July 2020 / Published: 4 August 2020
(This article belongs to the Special Issue Extracellular Vesicles: Biology and Potentials in Cancer Therapeutics)
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. View Full-Text
Keywords: extracellular vesicles; exosomes; small extracellular vesicles; isolation; purification; size-exclusion chromatography; ultracentrifugation; sucrose density cushion; lymph node; spleen; solid tissue extracellular vesicles; exosomes; small extracellular vesicles; isolation; purification; size-exclusion chromatography; ultracentrifugation; sucrose density cushion; lymph node; spleen; solid tissue
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Bordas, M.; Genard, G.; Ohl, S.; Nessling, M.; Richter, K.; Roider, T.; Dietrich, S.; Maaß, K.K.; Seiffert, M. Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues. Int. J. Mol. Sci. 2020, 21, 5586.

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