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Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications

Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV sepa...

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Autores principales: Tzaridis, Theophilos, Bachurski, Daniel, Liu, Shu, Surmann, Kristin, Babatz, Felix, Gesell Salazar, Manuela, Völker, Uwe, Hallek, Michael, Herrlinger, Ulrich, Vorberg, Ina, Coch, Christoph, Reiners, Katrin S., Hartmann, Gunther
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8431127/
https://www.ncbi.nlm.nih.gov/pubmed/34502122
http://dx.doi.org/10.3390/ijms22179211
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author Tzaridis, Theophilos
Bachurski, Daniel
Liu, Shu
Surmann, Kristin
Babatz, Felix
Gesell Salazar, Manuela
Völker, Uwe
Hallek, Michael
Herrlinger, Ulrich
Vorberg, Ina
Coch, Christoph
Reiners, Katrin S.
Hartmann, Gunther
author_facet Tzaridis, Theophilos
Bachurski, Daniel
Liu, Shu
Surmann, Kristin
Babatz, Felix
Gesell Salazar, Manuela
Völker, Uwe
Hallek, Michael
Herrlinger, Ulrich
Vorberg, Ina
Coch, Christoph
Reiners, Katrin S.
Hartmann, Gunther
author_sort Tzaridis, Theophilos
collection PubMed
description Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma.
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spelling pubmed-84311272021-09-11 Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications Tzaridis, Theophilos Bachurski, Daniel Liu, Shu Surmann, Kristin Babatz, Felix Gesell Salazar, Manuela Völker, Uwe Hallek, Michael Herrlinger, Ulrich Vorberg, Ina Coch, Christoph Reiners, Katrin S. Hartmann, Gunther Int J Mol Sci Article Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma. MDPI 2021-08-26 /pmc/articles/PMC8431127/ /pubmed/34502122 http://dx.doi.org/10.3390/ijms22179211 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Tzaridis, Theophilos
Bachurski, Daniel
Liu, Shu
Surmann, Kristin
Babatz, Felix
Gesell Salazar, Manuela
Völker, Uwe
Hallek, Michael
Herrlinger, Ulrich
Vorberg, Ina
Coch, Christoph
Reiners, Katrin S.
Hartmann, Gunther
Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications
title Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications
title_full Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications
title_fullStr Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications
title_full_unstemmed Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications
title_short Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications
title_sort extracellular vesicle separation techniques impact results from human blood samples: considerations for diagnostic applications
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8431127/
https://www.ncbi.nlm.nih.gov/pubmed/34502122
http://dx.doi.org/10.3390/ijms22179211
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