Fully Automated, Label-Free Isolation of Extracellular Vesicles from Whole Blood for Cancer Diagnosis and Monitoring

Abstract

Extracellular vesicles (EVs) that circulate in body fluids possess significant potential for disease diagnosis. Their use in clinical settings, however, has been limited owing to lack of simple and robust isolation methods. To rectify this problem, a centrifugal device for automatic, fast, and efficient isolation of EVs from whole-blood, called Exodisc-B is presented in this paper. Methods: The device comprises a built-in chamber to facilitate plasma separation and two nanoporous filters—one for removing larger particles and the other for enriching EVs. The performance of the device in comparison to ultracentrifugation (UC) was evaluated by analyzing the yield, purity, protein and RNA content of the isolated EVs. Additionally, the EV protein marker expressions were measured by ELISA and statistically analyzed to differentiate prostate cancer patients from healthy donors. Results: Compared with the UC technique, the proposed device is capable of isolating at least an order of magnitude higher number of EVs with about 30-fold higher mRNA count within 40 min. Sandwich ELISA of EV-specific membrane proteins—CD9-CD81—confirmed that Exodisc-B can isolate EVs from a volume of whole blood as low as 30 µL with a capture efficiency exceeding 75%. The device also facilitates temporal monitoring of tumor progression within live mouse xenograft models over a period of 13 weeks while using minimal volumes of weekly collected blood samples. Further, in ELISA analyses of multiple cancer-related proteins, such as prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor 1 (EGFR1), and heat shock protein 90 (HSP90), extracted from EVs isolated from human plasma, 43 patients were differentiated from 30 healthy donors. Conclusion: The results demonstrated the ability of Exodisc-B to provide a rapid, sensitive, and point-of-care-type method for extracting intact EVs from small volumes of clinical blood samples for disease diagnosis and monitoring.

Considerations and Implications in the Purification of Extracellular Vesicles – A Cautionary Tale

“…To address this issue, alternative methods for EV purification have been exploited. These include precipitation-based strategies; some of which can be coupled with antibody-capture methods on microfluidic devices or the use of magnetic nanowires (Ghosh et al, 2014; Kanwar et al, 2014; Lim et al, 2019; Sunkara et al, 2019). However, these methods may not be scalable, often compromise on EV purity (Yamada et al, 2012) and require more studies on if the purified EVs can be efficiently eluted and remain complementary to use for any downstream functional characterization.…”

MicroRNAs from Liquid Biopsy Derived Extracellular Vesicles: Recent Advances in Detection and Characterization Methods

“…While the problem of specimen has been addressed, we underlined the utility of technical adaptations of classic methods, such as ddPCR or fluorescent-based detection methods as they either provide quicker analysis [ 127 ] or feasibility for high-throughput screening [ 107 ] and require a smaller screening volume. If these techniques are further improved and the advantages combined, they might make implementing EV isolation in clinical settings possible.…”

“…Several studies are applying different techniques. Assays that are merely size based mainly consist of two filters of different sizes to discard cell debris in the first step and protein in the second step of filtration [127,128]. Another method traps EVs between micropillars with silicon nanowires [129].…”

Detection of EGFR Mutations Using Bronchial Washing-Derived Extracellular Vesicles in Patients with Non-Small-Cell Lung Carcinoma

“…EVs were isolated using a previously described lab-on-a-disc platform, Exo-Disc [ 41 , 43 , 44 ]. Each disc is composed of 6 sample chambers each of which is connected to an AAO membrane with 20 nm pore size for highly efficient tangential flow filtration.…”