Dataset Information

Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles.

ABSTRACT:

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Nanosized cell-derived membrane vesicles are increasingly recognized as therapeutic vehicles and high-potential biomarkers for several diseases. Currently available methods allow bulk analysis of vesicles but are not suited for accurate quantification and fail to reveal phenotypic heterogeneity in membrane vesicle populations. For such analyses, single vesicle-based, multiparameter, high-throughput methods are needed. We developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized membrane vesicles. Proof of principle was obtained by single-particle analysis of virions and liposomes. Further validation was obtained by quantification of cell-derived nanosized membrane vesicles from cell cultures and body fluids. An important aspect was that the technology was extended to detect specific proteins on individual vesicles. This allowed identification of exosome subsets and phenotyping of individual exosomes produced by dendritic cells (DCs) undergoing different modes of activation. The described technology allows quantitative, multiparameter, and high-throughput analysis of a wide variety of nanosized particles and has broad applications.

From the clinical editor

The authors developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized cell-derived membrane vesicles that are increasingly recognized both as therapeutic vehicles and high-potential biomarkers for several diseases. A high throughput, easily available, and sensitive detection method such as the one discussed here is a critically important prerequisite for further refinements of this technology.

SUBMITTER: Nolte-'t Hoen EN

PROVIDER: S-EPMC7106164 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles.

Nolte-'t Hoen Esther N M EN van der Vlist Els J EJ Aalberts Marian M Mertens Hendrik C H HC Bosch Berend Jan BJ Bartelink Willem W Mastrobattista Enrico E van Gaal Ethlinn V B EV Stoorvogel Willem W Arkesteijn Ger J A GJ Wauben Marca H M MH

Nanomedicine : nanotechnology, biology, and medicine 20111022 5

PMID: 22024193

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Project description:The cell-derived vesicles (CDVs) obtained using a proprietary extrusion process are the foundation of BioDrone platform technology. With superior productivity and versatility, this technology has garnered increasing attention in broad applications, particularly as a drug delivery vehicle. Previously, we showed that CDVs exhibited varying levels of expression for tetraspanin and organelle membrane markers while revealing no discernible differences in physical characteristics compared to naturally produced extracellular vesicles (EVs). To further understand and utilize the therapeutic potentials of CDVs, a more comprehensive study of membrane protein profiles is necessary. In addition, it is crucial to validate that the CDVs produced from extrusion are indeed intact lipid vesicles rather than other impurities. Here, we produced multiple batches of CDVs and EVs from HEK293 cells. CDVs and EVs were subjected to the same purification processes for subsequent proteome and particle analyses. The proteome analyses revealed unique proteome signatures between CDVs, EVs, and parental cells. Extensive proteome analyses identified the nine most prominent membrane markers that are abundant in CDVs compared to cells and EVs. Subsequent western blotting and nanoparticle flow cytometry analyses confirmed that CD63, lysosome-associated membrane glycoprotein 1 (LAMP1), and nicastrin (NCSTN) are highly enriched in CDVs, whereas CD81, CD9, and prostaglandin F2 receptor negative regulator (PTGFRN) are more abundant in EVs. This highlights the unique membrane composition and marker signature of CDVs that are distinct from EVs. Lastly, we demonstrated that more than 90% of the CDVs are genuine lipid vesicles by combining two different classes of vesicle labeling dyes and detergents to disrupt lipid membranes. This indicates that our proprietary extrusion technology is highly compatible with other well-characterized EV production methods. The robust CDV markers identified in this study will also facilitate the engineering of CDVs to achieve enhanced therapeutic effects or tissue-selective cargo delivery.

Dataset Information

Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles.

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From the clinical editor

Publications

Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles.

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