NEW YORK (360Dx) – The National University of Singapore is developing a platform that uses a table-top centrifuge and microfluidic chip to quickly isolate extracellular phospholipid vesicles that could be carriers of disease biomarkers.
In the future, the platform could become a valuable way to isolate extracellular vesicles suitable for diagnostic testing for cancers and other diseases through PCR, sequencing, or other methods, said Chwee Teck Lim, a developer of the technique and a professor in the department of biomedical engineering at the National University of Singapore.
The technique enables the isolation and capture of extracellular vesicles, consisting of microvesicles and exosomes, without using the antibody labels or expensive centrifuges that are currently used by researchers, Lim said.
The Singapore university team recently described the development and operation of their platform, called µCENSE (microfluidic CEntrifugal Nanoparticles Separation and Extraction), in the journal Biomicrofluidics.
Extracellular vesicles thought to be present in all body fluids are phospholipid spheres between 50 nm and 1000 nm in diameter. Shed by cells, their molecular make-up reflects that of their cell of origin. Researchers working in this area of diagnostics believe that it might be easier to measure proteins or nucleic acids in exosomes derived from, for instance, cancer cells, than to detect cancer-linked nucleic acids or protein circulating freely in a patient's blood or urine.
"The method developed by the [National University of Singapore] group is simple, rapid, and effective," said Tony Jun Huang, an exosome-based diagnostic platform developer and professor at Duke University's Pratt School of Engineering. "It could be very useful for exosome-based fundamental research or diagnosis.”
Jiashu Sun, a principal investigator of microfluidics at the National Center for Nanoscience and Technology in Beijing, noted that conventional techniques for exosome extraction often require large sample volumes, long processing times, and sophisticated instrumentation.
The work by the Singapore researchers reduces processing time by more than 100-fold compared to conventional methods, she said, adding that the research "presents a centrifugal microfluidic device for label-free, high-throughput, high-efficient, minimally damaged, and cost-effective isolation of exosomes, which is of great significance for downstream diagnostics."
Some of the main benefits of the new technique are derived from eliminating the use of antibodies to find disease markers, Lim said. "By using antibodies, you might capture a subpopulation but not all of the cancer cells or extracellular vesicles that you want to capture," he said.
Further, the new platform's ability to capture extracellular vesicles by combining a benchtop centrifuge with a microfluidic chip separates the nanoparticles into specific sizes and leads to high efficiency and high extraction purity, Lim said.
In the internal study, the table-top centrifuge achieved a "high separation efficiency" of 90 percent and an extraction purity of 85 percent.
The platform successfully extracted extracellular vesicles in eight minutes using 10 mL of a sample cultured from two separate cell lines, the breast cancer cell line MCF-7 and the lung cancer cell line H1975. The researchers used immunoblotting to identify exosomal CD63 protein markers in the extracted microvesicles from the different cell lines
Lim noted that the researchers are building a second microfluidic chip that accommodates larger samples that could be needed when relatively few exosomes are present in the biological fluid.
The new platform controls the flow of nanoparticles in a stable manner, Lim said. It is speedy and simple enough that it could eventually be used with laboratory or point-of-care liquid biopsy tests, he said, adding, "We wanted to come up with a technique that doesn't require reagents and was also faster than current alternative techniques."
The researchers said that they subjected vesicles between 100 nm and 1,000 nm to a "gentle" and "unique fluid flow" without the use of a syringe, which is frequently needed to pump fluids through tiny channels in a microfluidic chip. After separating different-size vesicles into different regions in the chip, they drew them out of the chip using pipettes, Lim said.
Although the platform shows promise for use in a clinical diagnostic workflow, it must first prove itself in studies using clinical samples, he said. The researchers have only just begun testing the platform for its clinical utility, seeking to identify both RNA and protein markers of cancers, cardiovascular diseases, and infectious diseases from liquid biopsies.
Companies in recent years have commercialized precipitation kits, which are alternate exosome extraction tools, but these require additional reagents that can affect the yield and processing time, Lim added.
In a method most frequently employed by researchers, vesicles experience high centrifugal force. However, with this method, some of the vesicles fuse together, and it can alter their structural properties, hampering extraction yield and making processing for diagnostic purposes more difficult, Lim noted.
The process, ultracentrifugation, can take about eight hours to separate vesicles prior to measuring them for disease markers, making them inefficient for use in most clinical diagnostic applications, Lim said.
Because the extracellular nanoparticles are so small and have such high diffusion characteristics, researchers currently need to use those high centrifugal forces so that they can pull the particles to where they need to be, he said.
Huang said that more than half of researchers use ultracentrifugation for isolating exosomes. But while ultracentrifugation has proven to be an effective method to extract exosomes, it is time-consuming, expensive, and demanding because highly training personnel are needed, and it has limited purity and yield, he said.
He noted that in general extracellular vesicles, including exosomes, have been identified as potentially transformative for the diagnosis and prognosis of multiple diseases. These include cancer, neurodegenerative diseases such as Parkinson’s and Alzheimer’s, and diseases of the kidney, heart, liver, and placenta, he said.
Nonetheless, difficulties in the consistent isolation of exosomes have greatly limited their clinical utility, he added.
Similar to the National University of Singapore group, others have been exploring different approaches to more effectively isolating extracellular vesicles.
For example, Huang and his colleagues at Duke are working with the Massachusetts Institute of Technology, the Magee-Womens Research Institute at the University of Pittsburgh, and Nanyang Technological University to develop a point-of-care prototype that combines acoustics with microfluidics to separate the exosome particles from blood, accelerating the potential for future liquid biopsy tests that could detect a broad range of medical conditions.
Ulsan National Institute of Science & Technology (UNIST) in South Korea is developing a liquid biopsy-based lab-on-a-disc that incorporates centrifugal force and nanoporous membranes in isolating and identifying extracellular vesicles that carry potentially important biomarkers of cancer in urine. The device provides EV separation within 30 minutes using a table-top, low G-force centrifuge.
Waltham, Massachusetts-based Exosome Diagnostics has already taken exosome technology to clinical use and begun marketing tests for lung and prostate cancer. In 2016, the firm launched its ExoDx Prostate IntelliScore test (EPI) through the company’s CLIA-certified laboratory in Cambridge.
ExoDx Prostate measures three exosomal RNA markers in urine to assess the risk of high-grade prostate cancer in patients being considering for prostate biopsy. The firm's ExoDx Lung(ALK) test analyzes exosomal RNA in blood to look for EML4-ALK fusions used for guiding therapy in non-small cell lung cancer.
All going according to plan, the Singapore researchers will pursue commercialization of their extracellular vesicle isolation platform, possibly through Clearbridge Biomedics, a company founded by Lim that's providing diagnostic platforms for research into circulating tumor cells.
Sun noted that for the Singapore researchers to be able to commercialize their platform, additional "efforts may be needed to evaluate its performance for extraction of exosomes in real clinical samples, such as human serum, as well as integrating a downstream platform for disease diagnostics."