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Theranostics 2019; 9(7):1851-1863. doi:10.7150/thno.32438 This issue Cite

Research Paper

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

Vijaya Sunkara^1,2*, Chi-Ju Kim^1,2*, Juhee Park², Hyun-Kyung Woo^1,2, Dongyoung Kim², Hong Koo Ha³, Mi-Hyun Kim⁴, Youlim Son⁵, Jae-Ryong Kim⁵, Yoon-Kyoung Cho^1,2✉

1. Department of Biomedical Engineering, School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea;
2. Center for Soft and Living Matter, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea;
3. Department of Urology, Pusan National University Hospital, Pusan National University School of Medicine, Busan 49241, Republic of Korea;
4. Department of Internal Medicine, Pusan National University Hospital, 179, Gudeok-ro, Seo-Gu, Busan 49241, Republic of Korea;
5. Department of Biochemistry and Molecular Biology, Smart-aging Convergence Research Center, College of Medicine, Yeungnam University, Daegu 42415, Republic of Korea.
*These authors contributed equally to this work.

✉ Corresponding author: E-mail: ykchoac.kr

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.

Keywords: extracellular vesicles, liquid biopsy, lab-on-a-disc, size-based filtration, ELISA

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