ExoDisc for CCS
®
(ExoDisc D20)
The below compares the performance of an ExoDisc™ in comparison to two commonly used EV-isolation methods: Ultracentrifugation (UC) and Exo-spin™ (precipitation).
Starting with 1 mL Of LNCaP CCS (Cell Culture Supernatant), EVs were isolated using three different protocols and characterized by nanoparticle-tracking analysis (NTA), enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR).
ExoDisc provides >95% recovery of EVs and >100x higher concentrations of mRNA as compared to the UC method.
Images of Intact EVs
Scanning Electron Microscopy
(SEM)
The EVs isolated/concentrated are shown sitting on top of the nano filter of an ExoDisc-D20, which has pores with 20 nm diameter.
The EVs have a range of size and are not stuck inside the pores.
Transmission Electron Microscopy
(TEM)
The size of EVs is more verified and the intact shape of EVs is visible while EVs isolated by ultracentrifugation commonly have the shapes of squeezed and crushed spheres.
Super-resolution Structured Illumination Microscopy (SR-SIM)
The image shows particles with EV-specific surface protein markers with green Alexa dye in the isolated sample observed by super-resolution microscopy.
High Performance - high yield and concentration
NTA (Nanoparticle Tracking Analysis)
Compare the size distribution and concentration of EVs isolated by three different methods using the same input material (supernatant of centrifuged culture media of LnCAP, prostate cancer cell line).
An ExoDisc gives the highest yield and does not lose large-sized EVs compared to UC (ultracentrifugation) and Exo-spin (3rd party precipitation method).
ELISA CD9/CD81 (sandwich assay)
RT-qPCR (real time quantitative PCR)
Not all particles collected are EVs. The particles obtained from ExoDisc include the most amount of particles with EV-specific surface markers (CD9, CD81).
The cargo mRNAs were quantified by RT-qPCR. A popular housekeeping gene, GAPDH, is found abundantly as mRNA cargo inside the EVs. The EV-specific surface marker CD9's mRNA is also the same. The EVs contained the mRNAs of PSA, PSMA - the biomarkers of prostate cancer abundantly. In each case, ExoDisc gave the most amount (smallest CT values) than competitions.
Total RNA
Total cargo RNA amount measured by Agilent's LabChip Bioanalyzer for RNA, and the structural integrity of the RNAs over a broad range of sizes were confirmed using electrophoresis.
High Performance - high purity
The isolated EVs can be washed with your favorite buffer solutions.
Two times of repeated wash by 1 ml of PBS buffer are usually enough for high purity.
Each washing step washes away water-soluble proteins residing outside of EVs.
Comparative Advantages
References
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Woo, H.-K., Sunkara V. et al. Exodisc for Rapid, Size-Selective, and Efficient Isolation and Analysis of Nanoscale Extracellular Vesicles from Biological Samples. ACS Nano 11, 1360–1370 (2017). https://doi.org/10.1021/acsnano.6b06131
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Kim, D. et al. EV-Ident: Identifying Tumor-Specific Extracellular Vesicles by Size Fractionation and Single-Vesicle Analysis. Anal. Chem. 92, 6010–6018 (2020). https://doi.org/10.1021/acs.analchem.0c00285
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Park, J., Lee, C. et al. Detection of EGFR mutations using bronchial washing-derived extracellular vesicles in patients with non-small-cell lung carcinoma. Cancers 12, 2822 (2020). https://doi.org/10.3390/cancers12102822
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Kim, J. et al. Three-Dimensional Human Liver-Chip Emulating Pre-Metastatic Niche Formation by Breast Cancer-Derived Extracellular Vesicles. ACS Nano 14, 14971-14988 (2020). https://doi.org/10.1021/acsnano.0c04778
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Kim, C., Kuczler, M. D. et al. Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis. J. Vis. Exp. 180 e62836 (2021). https://doi.org/10.3791/62836
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Leung, J. et al. Isolation and Characterization of Extracellular Vesicles Through Orthogonal Approaches for the Development of Intraocular EV Therapy. Invest. Ophthalmol. Vis. Sci. 65, (2024). https://doi.org/10.1167/iovs.65.3.6