A simple, high-throughput method of protein and label removal from extracellular vesicle samples

Welsh, Joshua; Killingsworth, Bryce; Kepley, Julia; Traynor, Tim; McKinnon, Kathy; Savage, Jason E.; Appel, Deven; Aldape, Kenneth; Camphausen, Kevin; Berzofsky, Jay A.; Ivanov, Alexander R.; Ghiran, Ionita; Jones, Jennifer C.

doi:10.1039/d0nr07830a

Cited by 7 publications

(8 citation statements)

References 32 publications

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“…Flow cytometry is an optical technique that has demonstrated detection of vesicles down to ∼40 nm in specialised cases (Zhu et al., 2014 ) and ∼100 nm using many modern conventional cytometers by light scatter and fluorescence (Morales‐Kastresana et al., 2019 ; Sandau et al., 2020 ; Stoner et al., 2016 ; Welsh, Killingsworth, et al., 2021 ). Through calibration of data, flow cytometry has been demonstrated to be capable of characterising particle diameter (Stoner et al., 2016 ; Tian et al., 2020 ; van der Pol, de Rond, et al., 2018 ; Welsh, Horak, et al., 2020 ), epitope abundance (Gorgens et al., 2019 ; Welsh, Jones, et al., 2020 ), epitope density (Welsh, Jones, et al., 2020 ), effective refractive index (Pleet et al., 2023 ; van der Pol, de Rond, et al., 2018 ) and number concentration within standardised size ranges (van der Pol, Sturk, et al., 2018 ).…”

Section: Technique‐specific Reporting Considerations For Ev Character...mentioning

confidence: 99%

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Welsh,

Goberdhan,

O'Driscoll

et al. 2024

J of Extracellular Vesicle

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356

163

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Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year‐on‐year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non‐vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its ‘Minimal Information for Studies of Extracellular Vesicles’, which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

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Section: Technique‐specific Reporting Considerations For Ev Character...mentioning

confidence: 99%

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Welsh,

Goberdhan,

O'Driscoll

et al. 2024

J of Extracellular Vesicle

Self Cite

356

163

View full text Add to dashboard Cite

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“…1I ) when applied to plasma after SEC or DMC (to remove lipoproteins). As Capto Core 700 resin has recently been shown to remove dye when used in 96-well filter plates (46), we envision MMR Slurry being adaptable to a high-throughput format and well-suited for profiling EVs from clinical samples for biomarker discovery. We also optimized and validated an EV immuno-isolation protocol that works in both plasma and CSF with or without prior EV enrichment.…”

Section: Discussionmentioning

confidence: 99%

Identification of markers for the isolation of neuron-specific extracellular vesicles

Ter-Ovanesyan,

Whiteman,

Gilboa

et al. 2024

Preprint

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Extracellular vesicles (EVs) are released by all cells and contain RNA and protein from their cell of origin. EVs in biofluids could be used as diagnostic biomarkers to non-invasively report the state of inaccessible cells, such as neurons in the brain. As biofluids such as cerebrospinal fluid (CSF) and plasma contain EVs originating from many different cells, isolating cell type-specific EVs and measuring their cargo could help determine the state of specific cell types. Here, we demonstrate an approach aiming to immuno-isolate EVs from neurons based on neuron-derived protein surface markers. We first developed a framework to select transmembrane proteins suitable as neuron-specific EV markers based on gene expression and EV proteomics data. Leveraging a novel, high-purity EV isolation method we developed, we further cataloged the proteins present on EVs in human CSF and plasma. Using immunoassays against several of the predicted neuron-specific proteins, we confirmed one marker, NRXN3 as present on EVs in CSF and plasma by size exclusion chromatography (SEC) and density gradient centrifugation (DGC). Finally, we developed efficient EV immuno-isolation methods and applied them to isolate NRXN3+ EVs. Our study provides a general methodology for the isolation of cell-type specific EVs and paves the way for the use of neuron-derived EVs to study and diagnose neurological disease.

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“…When required, fluorescent EVs were labeled after isolation by 1 h of incubation at 37 °C with carboxyfluorescin diacetate succinimidyl ester (CFDA-SE). This latest, after passing the membranes, undergoes hydrolysis of the diacetic groups, thus coverting into the fluorescent state CFSE [ 28 , 29 ]. The fluorophore excess was removed by the use of centrifugal filters.…”

Section: Methodsmentioning

confidence: 99%

Thermoresponsive M1 macrophage-derived hybrid nanovesicles for improved in vivo tumor targeting

Barone

Zimbo

d’Avanzo

et al. 2023

Drug Deliv. and Transl. Res.

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Despite the efforts and advances done in the last few decades, cancer still remains one of the main leading causes of death worldwide. Nanomedicine and in particular extracellular vesicles are one of the most potent tools to improve the effectiveness of anticancer therapies. In these attempts, the aim of this work is to realize a hybrid nanosystem through the fusion between the M1 macrophages-derived extracellular vesicles (EVs-M1) and thermoresponsive liposomes, in order to obtain a drug delivery system able to exploit the intrinsic tumor targeting capability of immune cells reflected on EVs and thermoresponsiveness of synthetic nanovesicles. The obtained nanocarrier has been physicochemically characterized, and the hybridization process has been validated by cytofluorimetric analysis, while the thermoresponsiveness was in vitro confirmed through the use of a fluorescent probe. Tumor targeting features of hybrid nanovesicles were in vivo investigated on melanoma-induced mice model monitoring the accumulation in tumor site through live imaging and confirmed by cytofluorimetric analysis, showing higher targeting properties of hybrid nanosystem compared to both liposomes and native EVs. These promising results confirmed the ability of this nanosystem to combine the advantages of both nanotechnologies, also highlighting their potential use as effective and safe personalized anticancer nanomedicine. Graphical Abstract

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A simple, high-throughput method of protein and label removal from extracellular vesicle samples

Abstract: Proposed pipeline to increase of the clinical utility extracellular vesicles (EVs) as translational biomarkers.

Cited by 7 publications

References 32 publications

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Identification of markers for the isolation of neuron-specific extracellular vesicles

Thermoresponsive M1 macrophage-derived hybrid nanovesicles for improved in vivo tumor targeting

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