Single extracellular vesicle analysis performed by imaging flow cytometry in contrast to NTA rigorously assesses the accuracy of urinary extracellular vesicle preparation techniques

Droste, Marvin; Tertel, Tobias; Jeruschke, Stefanie; Dittrich, Robin; Kontopoulou, Evangelia; Walkenfort, Bernd; Börger,; Pf, Hoyer; Ak, Büscher; Thakur, Binay; Giebel, Bernd

doi:10.1101/2021.04.01.437817

Cited by 6 publications

(1 citation statement)

References 46 publications

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“…Upon analyzing sEV samples that had been prepared from void urine with different small scale sEV preparation methods with NTA, IFCM and Western blot, we learned that IFCM data for the detection of CD9 + sEVs correlated well with semiquantitative Western blot data for the exosomal marker protein TSG101, but not with particle numbers recorded by traditional NTA. The NTA data instead correlated with Western blot intensities of uromodulin (Tamm Horsfall protein), an impurity marker in the urine-EV field [56].…”

Section: Strategies For the Upstream Processingmentioning

confidence: 99%

Scaled preparation of extracellular vesicles from conditioned media

Staubach

Bauer

Tertel

et al. 2021

Advanced Drug Delivery Reviews

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Section: Strategies For the Upstream Processingmentioning

confidence: 99%

Scaled preparation of extracellular vesicles from conditioned media

Staubach

Bauer

Tertel

et al. 2021

Advanced Drug Delivery Reviews

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Imaging flow cytometry challenges the usefulness of classically used EV labelling dyes and qualifies that of a novel dye, named Exoria™ for the labelling of MSC-EV preparations

Tertel

Schoppet²,

Stambouli

et al. 2021

Preprint

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Extracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is central for mediating the EVs role in intercellular communication processes, labelling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of the EVs target cells and tissues. However, the accuracy and specificity of commonly utilized labelling dyes has not been sufficiently analyzed. By combining recent advancements in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant MSC-EV preparations, we explored the EV labelling efficacy of various fluorescent dyes, specifically of CFDA-SE, Calcein AM, PKH67, BODIPY-TR-Ceramide and a novel lipid dye named Exoria. Our analyses qualified Exoria as the only dye which specifically labels EVs within our MSC-EV preparations. Furthermore, we demonstrate Exoria labelling does not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labelled EVs were differentially taken-up by different immune cell types. Overall, our results qualify Exoria as an appropriate dye for the labelling of EVs derived from our MSC-EV preparations, this study also demonstrates the need for the development of next generation EV characterization tools which are able to localize and confirm specificity of EV labelling.

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Detailed Characterization of Small Extracellular Vesicles from Different Cell Types Based on Tetraspanin Composition by ExoView R100 Platform

Breitwieser

Koch

Tertel

et al. 2022

IJMS

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Small extracellular vesicles (sEV) hold enormous potential as biomarkers, drug carriers, and therapeutic agents. However, due to previous limitations in the phenotypic characterization of sEV at the single vesicle level, knowledge of cell type-specific sEV signatures remains sparse. With the introduction of next-generation sEV analysis devices, such as the single-particle interferometric reflectance imaging sensor (SP-IRIS)-based ExoView R100 platform, single sEV analyses are now possible. While the tetraspanins CD9, CD63, and CD81 were generally considered pan-sEV markers, it became clear that sEV of different cell types contain several combinations and amounts of these proteins on their surfaces. To gain better insight into the complexity and heterogeneity of sEV, we used the ExoView R100 platform to analyze the CD9/CD63/CD81 phenotype of sEV released by different cell types at a single sEV level. We demonstrated that these surface markers are sufficient to distinguish cell-type-specific sEV phenotypes. Furthermore, we recognized that tetraspanin composition in some sEV populations does not follow a random pattern. Notably, the tetraspanin distribution of sEV derived from mesenchymal stem cells (MSCs) alters depending on cell culture conditions. Overall, our data provide an overview of the cell-specific characteristics of sEV populations, which will increase the understanding of sEV physiology and improve the development of new sEV-based therapeutic approaches.

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Single extracellular vesicle analysis performed by imaging flow cytometry in contrast to NTA rigorously assesses the accuracy of urinary extracellular vesicle preparation techniques

Cited by 6 publications

References 46 publications

Scaled preparation of extracellular vesicles from conditioned media

Scaled preparation of extracellular vesicles from conditioned media

Imaging flow cytometry challenges the usefulness of classically used EV labelling dyes and qualifies that of a novel dye, named Exoria™ for the labelling of MSC-EV preparations

Detailed Characterization of Small Extracellular Vesicles from Different Cell Types Based on Tetraspanin Composition by ExoView R100 Platform

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