Exodisc for Rapid, Size-Selective, and Efficient Isolation and Analysis of Nanoscale Extracellular Vesicles from Biological Samples

Abstract

Extracellular vesicles (EVs) are cell-derived, nanoscale vesicles that carry nucleic acids and proteins from their cells of origin and show great potential as biomarkers for many diseases, including cancer. Efficient isolation and detection methods are prerequisites for exploiting their use in clinical settings and understanding their physiological functions. Here, we presented a rapid, label-free, and highly sensitive method for EV isolation and quantification using a lab-on-a-disc integrated with two nanofilters (Exodisc). Starting from raw biological samples, such as cell-culture supernatant (CCS) or cancer-patient urine, fully automated enrichment of EVs in the size range of 20-600 nm was achieved within 30 min using a tabletop-sized centrifugal microfluidic system. Quantitative tests using nanoparticle-tracking analysis confirmed that the Exodisc enabled >95% recovery of EVs from CCS. Additionally, analysis of mRNA retrieved from EVs revealed that the Exodisc provided >100-fold higher concentration of mRNA as compared with the gold-standard ultracentrifugation method. Furthermore, on-disc enzyme-linked immunosorbent assay using urinary EVs isolated from bladder cancer patients showed high levels of CD9 and CD81 expression, suggesting that this method may be potentially useful in clinical settings to test urinary EV-based biomarkers for cancer diagnostics.

Microfluidics for exosome isolation and analysis: enabling liquid biopsy for personalized medicine

“…In an expected advancement, two integrated filtration -based platforms have been recently developed for analysis of EVs and, interestingly, they both feature a double filtration approach. 84,85 Liang et al . employed a 200-nm pore size membrane to retain larger EVs and impurities from urine samples, while isolating and enriching small EVs by means of a second membrane with a pore size of 30 nm that allows proteins to pass through.…”

Breast Cancer Recurrence Risk Assessment: Is Non-Invasive Monitoring an Option?

“…Furthermore, the urinary proteome is known to be less contaminated by high-abundant proteins as is the case with blood, where only 22 different proteins account for 99% of an analyzed aliquot, masking potential biomarkers and making additional cleaning steps essential [47, 57]. Additionally, urine is seen to be more stable as compared to blood, where more proteolytic activity can be measured [63]. …”

“…A variety of targets in serum itself may be examined in order to measure metastatic cancer cell dissemination, including proteins, CTCs, cfDNA, miRNAs, and extracellular vesicles [63-67]. …”

Microdevices for Non-Invasive Detection of Bladder Cancer

“…The ease of obtaining patient urine samples facilitates testing and development of novel devices, but also provides ample material to detect and isolate materials indicating the presence of cancerous cells. While some screening and diagnostic devices target biomolecules or proteins, others isolate exfoliated tumor cells [80] and nucleic acid-containing vesicles [81]; this genetic material could provide a starting point for downstream genetic analyses to inform treatment decisions and provide insights into bladder cancer development and variants without requiring expensive and invasive biopsy procedures. In studies on lung [126] and breast cancer [127], microfluidic devices for mimicking tumor microenvironments provide a path toward tailored treatment approaches, while single-cell 'omics' analysis yields invaluable insight into the heterogeneity and molecular processes of cancer [128].…”