ExoDisc for
Plasma and Serum
®
(ExoDisc D100)
Plasma and serum are liquid portions of blood and they contain ~10¹² EVs per ml.
The EVs in blood have protein corona which makes the EV behave as if they are much larger than reality (increase of hydrodynamic radii).
ExoDisc-D100 for blood EVs, therefore, has larger-sized pores (pore-diameter 100 nm), and the rotational profile is different from ExoDisc-D20 (for CCS and urine).
The TFF function is also excellent, but up to 100 µl or 200 µl of plasma can be applied at a time by diluting with PBS buffer to enhance fluidity.
High Performance
NTA (Nanoparticle Tracking Analysis)
ELISA CD9/CD81 (sandwich assay)
BCA
The NTA results are scalable from 10 µl up to 200 µl, and sandwich ELISA (CD9/CD81) is also very linear which means the quality of EV isolation does not deteriorate which is not possible with conventional TFF with low volume.
The linearly increasing BCA results proportional to the input plasma volume are obtained from washed blood EVs (only cargo protein was quantified in this data).
Total RNA
RT-qPCR
ELISA/Western Blotting
LabChip (Bioanalyzer; RNA electrophoresis) data shows the RNA amount of EVs from ExoDisc is much more abundant and the large mRNAs stay intact compared to ultracentrigutation (UC) method.
The cargo mRNA were quantified by RT-qPCR. Popular house keeping gene GAPDH mRNAs are found in the EVs, and the EV-specific surface markers (CD9, CD63, CD81)'s mRNA are found inside EVs. ExoDisc found the most amount (smallest CT values) in each case.
Albumin which is abundant in blood and an indicator of contamination is minimal in ExoDisc compared to UC. The same was confirmed with western blotting.
Application:
Monitoring Tumor Growth in Live Mouse Xenograft Models
Usual methods for blood EV isolation and analysis require a large amount of blood samples and mice have to be sacrificed.
In contrast, ExoDisc has so high yield that it is possible to get only 100 µl of blood repeatedly every week from the same mouse.
Weekly monitoring was performed and the prostate tumor markers such as PSMA, EGFR, HSP90 increased as the innoculated prostate cancer cell line grew (tumor volume increase).
Application:
Protein Analysis of EVs from Cancer Patients' Plasma
The obtained EVs were used for microfluidic protein analysis and showed significant changes in tumor marker expression levels.
References
-
Sunkara, V., Kim, C.J. et al. Fully Automated, Label-Free Isolation of Extracellular Vesicles from Whole Blood for Cancer Diagnosis and Monitoring. Theranostics 9, 1851-1863 (2019). https://doi.org/10.7150/thno.32438
-
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
-
Dong, L., Zieren, R. C., Horie, K. et al. Comprehensive Evaluation of Methods for Small Extracellular Vesicles Separation from Human Plasma, Urine and Cell Culture Medium. J Extracell Vesicles (2020). https://doi.org/10.1002/jev2.12044