Universität zu Köln

Tzaridis, Theophilos ORCID: 0000-0001-9651-1144, Bachurski, Daniel ORCID: 0000-0001-9168-9680, Liu, Shu, Surmann, Kristin, Babatz, Felix, Salazar, Manuela Gesell, Voelker, Uwe, Hallek, Michael, Herrlinger, Ulrich, Vorberg, Ina ORCID: 0000-0003-0583-4015, Coch, Christoph, Reiners, Katrin S. and Hartmann, Gunther (2021). Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications. Int. J. Mol. Sci., 22 (17). BASEL: MDPI. ISSN 1422-0067

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Abstract

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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Tzaridis, Theophilos UNSPECIFIED orcid.org/0000-0001-9651-1144 UNSPECIFIED Bachurski, Daniel UNSPECIFIED orcid.org/0000-0001-9168-9680 UNSPECIFIED Liu, Shu UNSPECIFIED UNSPECIFIED UNSPECIFIED Surmann, Kristin UNSPECIFIED UNSPECIFIED UNSPECIFIED Babatz, Felix UNSPECIFIED UNSPECIFIED UNSPECIFIED Salazar, Manuela Gesell UNSPECIFIED UNSPECIFIED UNSPECIFIED Voelker, Uwe UNSPECIFIED UNSPECIFIED UNSPECIFIED Hallek, Michael UNSPECIFIED UNSPECIFIED UNSPECIFIED Herrlinger, Ulrich UNSPECIFIED UNSPECIFIED UNSPECIFIED Vorberg, Ina UNSPECIFIED orcid.org/0000-0003-0583-4015 UNSPECIFIED Coch, Christoph UNSPECIFIED UNSPECIFIED UNSPECIFIED Reiners, Katrin S. UNSPECIFIED UNSPECIFIED UNSPECIFIED Hartmann, Gunther UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-593489
DOI:	10.3390/ijms22179211
Journal or Publication Title:	Int. J. Mol. Sci.
Volume:	22
Number:	17
Date:	2021
Publisher:	MDPI
Place of Publication:	BASEL
ISSN:	1422-0067
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language MEMBRANE-VESICLES; ULTRACENTRIFUGATION; CHROMATOGRAPHY; PROTEINS; PRION; RNA Multiple languages Biochemistry & Molecular Biology; Chemistry, Multidisciplinary Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/59348