Dataset Information

Extracellular vesicle isolation and counting system (EVics) based on simultaneous tandem tangential flow filtration and large field-of-view light scattering.

ABSTRACT: Although the isolation and counting of small extracellular vesicles (sEVs) are essential steps in sEV research, an integrated method with scalability and efficiency has not been developed. Here, we present a scalable and ready-to-use extracellular vesicle (EV) isolation and counting system (EVics) that simultaneously allows isolation and counting in one system. This novel system consists of (i) EVi, a simultaneous tandem tangential flow filtration (TFF)-based EV isolation component by applying two different pore-size TFF filters, and (ii) EVc, an EV counting component using light scattering that captures a large field-of-view (FOV). EVi efficiently isolated 50-200 nm-size sEVs from 15 µL to 2 L samples, outperforming the current state-of-the-art devices in purity and speed. EVc with a large FOV efficiently counted isolated sEVs. EVics enabled early observations of sEV secretion in various cell lines and reduced the cost of evaluating the inhibitory effect of sEV inhibitors by 20-fold. Using EVics, sEVs concentrations and sEV PD-L1 were monitored in a 23-day cancer mouse model, and 160 clinical samples were prepared and successfully applied to diagnosis. These results demonstrate that EVics could become an innovative system for novel findings in basic and applied studies in sEV research.

SUBMITTER: Bae JH

PROVIDER: S-EPMC11231039 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Extracellular vesicle isolation and counting system (EVics) based on simultaneous tandem tangential flow filtration and large field-of-view light scattering.

Bae Ju-Hyun JH Lee Chan-Hyeong CH Jung Dokyung D Yea Kyungmoo K Song Byoung-Joon BJ Lee Hakho H Baek Moon-Chang MC

Journal of extracellular vesicles 20240701 7

Although the isolation and counting of small extracellular vesicles (sEVs) are essential steps in sEV research, an integrated method with scalability and efficiency has not been developed. Here, we present a scalable and ready-to-use extracellular vesicle (EV) isolation and counting system (EVics) that simultaneously allows isolation and counting in one system. This novel system consists of (i) EVi, a simultaneous tandem tangential flow filtration (TFF)-based EV isolation component by applying t ...[more]

PMID: 38978321

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Project description:ObjectiveThe aim of the current study was, first, to assess the coronary artery calcium (CAC) scoring potential of spectral photon-counting CT (SPCCT) in comparison with computed tomography (CT) for routine clinical protocols. Second, improved CAC detection and quantification at reduced slice thickness were assessed.MethodsRaw data was acquired and reconstructed with several combinations of reduced slice thickness and increasing strengths of iterative reconstruction (IR) for both CT systems with routine clinical CAC protocols for CT. Two CAC-containing cylindrical inserts, consisting of CAC of different densities and sizes, were placed in an anthropomorphic phantom. A specific CAC was detectable when 3 or more connected voxels exceeded the CAC scoring threshold of 130 Hounsfield units (HU). For all reconstructions, total CAC detectability was compared between both CT systems. Significant differences in CAC quantification (Agatston and volume scores) were assessed with Mann-Whitney U tests. Furthermore, volume scores were compared with the known CAC physical.ResultsCAC scores for routine clinical protocols were comparable between SPCCT and CT. SPCCT showed 34% and 4% higher detectability of CAC for the small and large phantom, respectively. At reduced slice thickness, CAC detection increased by 142% and 169% for CT and SPCCT, respectively. In comparison with CT, volume scores from SPCCT were more comparable with the physical volume of the CAC.ConclusionCAC scores using routine clinical protocols are comparable between conventional CT and SPCCT. The increased spatial resolution of SPCCT allows for increased detectability and more accurate CAC volume estimation.Key points• Coronary artery calcium scores using routine clinical protocols are comparable between conventional CT and spectral photon-counting CT. • In comparison with conventional CT, increased coronary artery calcium detectability was shown for spectral photon-counting CT due to increased spatial resolution. • Volumes scores were more accurately determined with spectral photon-counting CT.

Project description:Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.

Dataset Information

Extracellular vesicle isolation and counting system (EVics) based on simultaneous tandem tangential flow filtration and large field-of-view light scattering.

Publications

Extracellular vesicle isolation and counting system (EVics) based on simultaneous tandem tangential flow filtration and large field-of-view light scattering.

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