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Acoustics-Based Method for Isolating Exosomes Could Accelerate Liquid Biopsies


NEW YORK (360Dx) – Researchers have developed a point-of-care prototype that combines acoustics with microfluidics to separate tiny exosome particles from blood, accelerating the potential for future liquid biopsy tests that could detect a broad range of medical conditions.

Its developers at Duke University, the Massachusetts Institute of Technology, the Magee-Womens Research Institute at the University of Pittsburgh, and Nanyang Technological University are developing the device for early detection of cancers, neurodegenerative diseases, cardiovascular diseases, kidney disease, and liver disease.

Their prototype leverages the property of human cells to secrete exosomes, which are nanoscale packets that carry molecules and that can signal the presence or absence of disease.

In a study published last week in the Proceedings of the National Academy of Sciences, the researchers describe a device that uses a combination of microfluidics and sound waves to isolate exosomes from blood. They hope to eventually use the technology as the basis for a portable device that could analyze patient blood without requiring lab processing and without requiring time-consuming methods currently used to isolate exosomes prior to their analysis as disease biomarkers.

The group is close to completing a production prototype that is ready for licensing for research use, and early next year, they should be ready to manufacture a system for research use, including biomarker discovery of exosomes and biomarker monitoring of cancer or placenta health, Tony Jun Huang, one of the study's senior authors and a professor of mechanical engineering and materials science at Duke University, said in an interview.

In 2012, Huang cofounded a company, Ascent Bio-Nano Technologies, to work on commercializing the system. The researchers said that they expect in the long term to see the platform applied as an in vitro diagnostic at the point of care. "However, we would need to prove the use of the technology in research," Huang said, "and as more people adopt it, obtaining regulatory approval to market the tool as a diagnostic device will become much easier."  

"The motivation to advance exosomal technologies for research is driven by exosomes’ increasing traction as reliable biomarkers," said Cesar Castro, director of the cancer program at MGH Center for Systems Biology. Castro's group at MGH is developing an exosome-based multiplexed nanoplasmonic assay that would fit into clinical workflows.

He noted that enthusiasm about exosomal technologies is tempered by the limitations in their workflows and the yield of existing approaches. "Oftentimes, laborious techniques and handling of biospecimens interfere with the purity and quality of derived exosomes for downstream analyses," he said.

Therefore, the technology developed by Huang and colleagues "reflects an important advance in the field of exosome research," Castro said. "The group, already well-known for its strengths in acoustic analyses, offers researchers a contact-free solution" that doesn't require the application of beads and "is basically hassle-free," he noted.

Ming Dao, director of the Nanomechanics Laboratory at MIT, said in a statement that centrifugation-free sorting of different blood components "can drastically reduce the cost and processing time involved with liquid biopsy assays."

Many research groups are investigating the biological functions of exosomes, but the effort to develop exosome-based diagnostic tests is quite recent, Yoon-Kyoung Cho, a professor of biomedical engineering at Ulsan National Institute of Science & Technology (UNIST), South Korea, recently told 360Dx.

Exosomes are almost perfect as a target for early diagnosis, Huang said, but researchers have been challenged with isolating them prior to measurement. In 2014, Huang and his colleagues reported that they could separate micrometer-sized cells from blood by exposing them to sound waves as they flowed through a tiny channel.

In their present research, they have demonstrated their abilities to isolate far smaller entities.  

Exosomes are usually about 30 to 150 nanometers in diameter and their miniscule size makes them difficult to study and challenging to separate from their native biological fluids. Other researchers use ultracentrifugation, a process that involves spinning samples in a centrifuge, but which captures only a small fraction of the nanoparticles present in a biological fluid, Huang said.

Centrifugal spinning leads to gravitational forces of around 200,000 Gs, and it can take from six hours to a few days to separate the exosomes from blood, Huang said. That can damage the exosomes and produce lower yields than are desirable, he noted, adding that a ultracentrifuge setup is expensive and can cost around $100,000.

"Obviating the need for ultracentrifugation will not only prove a boon to existing investigators but also opens up avenues to new researchers and less endowed laboratories domestically and abroad," Castro said, adding, "Importantly, the technology’s ability to improve yield and purity aligns nicely with existing biobanks of precious clinical materials. Therefore, more can be done with less, [which is] a win-win for patients and researchers alike."

Huang noted that their microfluidic prototype is automated and allows single-step, on-chip isolation of exosomes from whole biological fluids with a high rate of purity and yield.

The device creates a high-frequency sound wave traveling at an angle to a sample liquid flowing through a tiny tube. By carefully tailoring the angle and frequency of the sound wave to the length of the channel and size of the particles, they can push particles larger than 1,000 nanometers into a separate channel.

This removes elements of blood, such as white blood cells, red blood cells, and platelets, the researchers said. The fluid then flows through a second chamber, where the same force is used to filter out everything smaller than 130 nanometers.

"Bioparticles of different size and with different physical properties experience dissimilar forces in response to the application of ultrasound," Huang said. "The particles, therefore, have different trajectories in the fluidic flow, and they flow to different outlets." That enables the researchers to isolate exosomes and differentiate them from other particles.

The dual-stage technique showed the ability to separate more than 80 percent of exosomes present with a purity of 98 percent, the researchers said, adding it took less than 25 minutes to process a 100-microliter undiluted blood sample. Further, they aim to reduce the exosome isolation time to less than 10 minutes, Huang said.

Subra Suresh, president-designate of Nanyang Technological University in Singapore and former president of Carnegie Mellon University, said in a statement that the research "provides a unique combination of microfluidics and acoustics, using state-of-the-art microfabrication technologies."

Huang noted that the team is developing a microfluidic chip that would be disposable and cost around $.25 when it's injection-molded at high volumes. A portable system, similar to the size of a laptop, which processes and displays the microfluidic chip's signals, would cost less than $3,000, he said.

In searching for noninvasive methods to do early diagnosis of cancers, researchers in the past few years have been developing liquid biopsy-based blood tests that avoid the challenges of doing direct biopsies, including risks associated with invasive procedures that can cause infections.

In developing liquid biopsy tests, researchers have been investigating detecting circulating tumor cells and, separately, cell-free DNA. Exosomes have an advantage over circulating tumor cells and cell-free DNA because they are more abundant, have a relatively long lifespan, and can be accessed from blood, urine, and cerebrospinal fluid, Huang said, adding that they are also very robust and stable, because they are "capable of protecting their content from the extracellular environment and preserving it throughout multiple freeze and thaw cycles."

Several research groups developing exosome-based methods are taking aim at eventually launching in vitro diagnostic tests for various diseases.

Castro's group at Harvard and MGH is developing an exosome-based multiplexed nanoplasmonic assay for high-throughput detection of pancreatic ductal adenocarcinoma, an aggressive and often inoperable form of pancreatic cancer.

Cambridge, Massachusetts-based Exosome Diagnostics has licensed the technology and is exploring integrating the assay with its portfolio of exosome tests to potentially perform concurrent testing of exosome-based proteins and mRNA.

Exosome Diagnostics has also licensed technology from a separate project also under development at Harvard and MGH. Hakho Lee's lab in the Center for Systems Biology is developing exosome-based diagnostic tests for several diseases.

Using both POC and high-volume tests, the group is investigating diagnostics for many types of cancers, including brain, colorectal, ovarian, pancreatic, and others.

With a view to developing a bladder cancer diagnostic test, Cho's group at UNIST is developing an alternative to traditional ultracentrifugation. It's lab-on-a-disc employs a table top, low G-force centrifuge to separate exosomes in urine within 30 minutes.

As a next step, Huang and his colleagues are working toward using acoustic waves to separate out even smaller nanoparticles such as viruses in a label-free, contact-less manner.

The researchers also have an ongoing project expected to continue until 2020 to look for markers related to abnormal pregnancy. The project is funded by a grant from the National Institutes of Health.

Factors to consider for future research and validation of the acoustic microfluidic device include the potential for activation of platelets by sound waves, Castro said. "Activated platelets may also shed many vesicles that may create unwanted noise," he noted.

Further clarity is needed on how Huang and his colleagues seek to scale up the technology to process large volume amounts for higher throughput, he said.