New Technologies to Enable Clinical Single Cell Analysis

Personalized medicine is driving the need for isolation of rarer target cell populations, including for the enrichment of circulating tumor cells (CTCs), hematopoietic stem cells, and circulating fetal cells from blood. Current molecular analysis, while increasing biological understanding of cancer and other diseases at the DNA level, often assesses “averaged” cellular populations—derived from millions of cells across a tumor population—rather than individual cells. But, scientists believe that hard-to-detect low-level “subclones” are really the drivers of relapse or disease progression.

A review of single-cell genomics in cancer, published in the Oct. 15, 2015 issue of Human Molecular Genetics, explains that ~1,000X sequencing (which is “way beyond” the depth seen in most studies) would be required to detect 99 percent of mutations carried by a one percent tumor-mass subclone analyzed at the bulk level, whereas with single-cell analysis only ~200 cells are required to reliably detect one percent tumor-mass clones.

“While bulk tissue genomic analysis across large populations of tumor cells has provided key insights into cancer biology, this approach does not provide the resolution that is critical for understanding the interaction between different genetic events within the cellular hierarchy of the tumor during disease initiation, evolution, relapse, and metastasis,” writes Quin Wills, D.Phil., from University of Oxford in the United Kingdom, in the review.

The challenge remains in isolating those 200 cells, in a manner that has high throughput, is affordable, and preserves the cell’s biological integrity. A flurry of activity advancing microfluidic technology to improve cell sorting is bringing the possibility of single cell analysis closer to a clinical reality.

Sorting Methodology Enables Further Analysis
Cell sorting is often used to enrich or purify cell samples into well-defined populations. Cell sorting serves as the first step in many diagnostic and therapeutic applications, such as the enrichment of hematopoietic stem cells for autologous patient treatments.

Experts predict that cell sorting on microfluidic devices will emerge as the platform for the next generation of cell sorters. Among the platform’s advantages are accessible fabrication, low reagent consumption, small footprints, and improved safety/contamination risk due to elimination of aerosols.

Lab-on-a-chip formats are emerging that are capable of cellular isolation and processing for downstream interrogation. These miniaturized formats enable sorting of cells based on a wider range of physical and molecular properties than standard flow cytometers, often without antibodies. However, in order for these technologies to achieve potential commercialization, experts say that several “significant” limitations must be overcome. In a review on microfluidics for cell separation, published Feb. 2015 in Lab on a Chip,

lead author C. Wyatt Shields, IV, from Duke University highlights some challenges including: scaling throughput and processing speeds to make processing clinical-scale samples (more than 500 million cells) feasible; ensuring operating pressures don’t impact cell function or viability; shrinking instrumentation footprints; reducing the level of technical expertise needed for machine operation; and reducing unit and sample processing costs for clinical application.

Commercialization
Despite the formidable challenges there is much excitement around single-cell genomic DNA analysis, particularly in cancer care, and proof-of-principle studies are beginning to emerge. Yet, to date, trials are small scale and the technology still has to meaningfully enter clinical practice. Experts say that while prototypes exist, the technology requires further investment to ensure affordability, manufacturability, and repeatable performance before commercialization is viable.

“The future of microfluidic cell sorting is promising,” writes Shields. “With the large variety of cell sorting devices … microfluidic cell sorting technologies can offer speeds and accuracies in a number of ways that rival current commercial devices in a platform that is more efficient, less cumbersome, and offers a more straightforward standard operating procedure.”

DTET spoke to two early-stage companies about their technologies and next steps to bring single cell sorting technologies to the commercial market.

“We think of it like a sandwich. If the meat is the sequencing, one bread is data analysis and the other bread is sample prep,” Jose Morachis, Ph.D., CEO of NanoCellect Biomedical (San Diego) tells DTET. “New [sample prep] technologies like ours are the exciting next frontier in sequencing.”

NanoCellect’s WolfCell Sorter platform is based upon microfluidic technology licensed from University of California, San Diego. The company believes the portable, bench-top platform addresses some of the current limitations and accessibility of flow cytometry technology. The platform integrates novel detection algorithms with on-chip sorting that uses a piezoacoustic actuator to gently direct cells into collection channels. The technology reduces shear stress on cells, while the disposable, closed chip eliminates dangerous aerosols and potentially brings flow cytometry abilities to more laboratories, the company says.

The integration of cell-sorting and single-cell positioning will allow diverse downstream applications—including kinetic analysis of secreted particles, genomic sequencing, gene expression, and imaging. Morachis says the company’s technology enables advancements in the CTC field, as well as potentially stem cell and CRISPR applications. The cell sorter is currently in beta testing, with a larger release of the $100,000 platform for the research market expected in the summer of 2016.

Liquid biopsies of CTCs are considered the low-hanging fruit of the single-cell analysis market. While evidence of clinical utility is still necessary to further adoption, experts say the application is already showing signs of “maturing.”

Silicon Biosystems (a subsidiary of the pharmaceutical company Menarini Group; Italy), however, has its eye set, not just on the liquid biopsy market, but also on capturing the market for solid tumor samples that are currently “discarded” by molecular labs for insufficient content of tumor cells—currently estimated to be as high as 20 percent to 30 percent of samples.

“We demonstrated we can recover it for NGS analysis even if the sample only has 5 percent tumor cells,” Raimo Tanzi, Silicon Biosystems’ chief commercial officer, tells DTET.

Molecular analysis from formalin-fixed paraffin-embedded (FFPE) samples characterizes all DNA present, including DNA from tumor cells, stromal cells, and infiltrating lymphocytes. Thus, the relative frequency of a given mutation in the heterogeneous mix of DNA molecules is all that can be assessed, while copy number aberration and other genomic instabilities are very difficult to detect, the company says. Its DEPArray system can be used to separate tumor cells from stromal, and other cells in the FFPE section and a “highly pure” collection of tumor cells can be recovered enabling complete genomic analysis.

The company’s DEPArray platform utilizes a semiconductor controlled flow chip where individual cells are coupled to single microelectrodes by dielectrophoretic (DEP) forces. The system combines the ability to manipulate individual cells with high quality image-based cell selection. The company says the technology identifies and sorts individual cells, or pools of cells, with 100 percent purity for detailed molecular analysis. Tanzi says the company will launch pure FFPE tumor analysis as a service at its San Diego-based CLIA lab later in the year.

New Technological Approaches In the last year many academic research groups published studies on novel sorting technologies. Below is a sampling.

• Parallel Microfiltration Method (PMF) is a screening method that utilizes cell squishiness to classify cancer cells. The researchers, from University of California, Los Angeles, say cancer cells are generally two to five times squishier than normal cells, with pliability similar to that of Jell-O. PMF enables simultaneous measurements of cell mechanotype across multiple samples by using uniform air pressure to drive cell suspensions through porous membranes. The relative “deformability” of a cell sample is quantified by the fraction of sample retained above the membrane, with stiffer cells blocking the pores and squishier cells allowing more of the celland- liquid mixture to pass through. The team found that drug-resistant human ovarian cancer cells are softer than their drug-sensitive counterparts, and that more-invasive cancer cells are softer than less-invasive ones. (The technology was described in a study published Dec. 2, 2015 in Scientific Reports.)

• Acoustic Separation Method (“Acoustic Tweezers”) works by using tilted-angle standing surface acoustic waves to apply pressure to a continuous flow of blood. Based on their size and weight, cancerous cells are forced out of the stream into a different channel, where they are collected. The power, intensity, and frequency used to generate the waves in this study are gentle (similar to common ultrasounds), the researchers say, helping to preserve the biocompatibility of the CTCs. The researchers, from Pennsylvania State University, report a throughput rate 20 times higher than previously achieved with similar devices, along with a separation rate of more than 83 percent. The researchers also say they found a way to separate the fluid-containing part of the device from the more expensive ultrasound-producing piezoelectric substrate, making “disposable” acoustic tweezers a cheap possibility. (The technology was described in a study published in April 2015 in the Proceedings of the National Academy of Sciences.)

• Microstructure-Constricted Filtration and Pneumatic Microvalves combine in this integrated microfluidic device to separate cells based on size and deformability. The researchers, from Northwest A&F University in China, say the technology can separate cancer cells from blood samples with more than 90 percent cell recovery and 80 percent purity. In a test of mesenchymal-like cancer cells and epithelium-like cancer cells (with different deformability) the technology demonstrated a “high” selectivity and recovery rate, the authors say. The high viability of the target cells benefits the ability to conduct downstream analysis. The device can also capture and release cells repeatedly, which improves its scalability. (The technology was described in the Aug. 2015 issue of The Analyst.)

Personalized medicine is driving the need for isolation of rarer target cell populations, including for the enrichment of circulating tumor cells (CTCs), hematopoietic stem cells, and circulating fetal cells from blood. Current molecular analysis, while increasing biological understanding of cancer and other diseases at the DNA level, often assesses “averaged” cellular populations—derived from millions of cells across a tumor population—rather than individual cells. But, scientists believe that hard-to-detect low-level “subclones” are really the drivers of relapse or disease progression.

A review of single-cell genomics in cancer, published in the Oct. 15, 2015 issue of Human Molecular Genetics, explains that ~1,000X sequencing (which is “way beyond” the depth seen in most studies) would be required to detect 99 percent of mutations carried by a one percent tumor-mass subclone analyzed at the bulk level, whereas with single-cell analysis only ~200 cells are required to reliably detect one percent tumor-mass clones.

“While bulk tissue genomic analysis across large populations of tumor cells has provided key insights into cancer biology, this approach does not provide the resolution that is critical for understanding the interaction between different genetic events within the cellular hierarchy of the tumor during disease initiation, evolution, relapse, and metastasis,” writes Quin Wills, D.Phil., from University of Oxford in the United Kingdom, in the review.

The challenge remains in isolating those 200 cells, in a manner that has high throughput, is affordable, and preserves the cell’s biological integrity. A flurry of activity advancing microfluidic technology to improve cell sorting is bringing the possibility of single cell analysis closer to a clinical reality.

Sorting Methodology Enables Further Analysis
Cell sorting is often used to enrich or purify cell samples into well-defined populations. Cell sorting serves as the first step in many diagnostic and therapeutic applications, such as the enrichment of hematopoietic stem cells for autologous patient treatments.

Experts predict that cell sorting on microfluidic devices will emerge as the platform for the next generation of cell sorters. Among the platform’s advantages are accessible fabrication, low reagent consumption, small footprints, and improved safety/contamination risk due to elimination of aerosols.

Lab-on-a-chip formats are emerging that are capable of cellular isolation and processing for downstream interrogation. These miniaturized formats enable sorting of cells based on a wider range of physical and molecular properties than standard flow cytometers, often without antibodies. However, in order for these technologies to achieve potential commercialization, experts say that several “significant” limitations must be overcome. In a review on microfluidics for cell separation, published Feb. 2015 in Lab on a Chip,

lead author C. Wyatt Shields, IV, from Duke University highlights some challenges including: scaling throughput and processing speeds to make processing clinical-scale samples (more than 500 million cells) feasible; ensuring operating pressures don’t impact cell function or viability; shrinking instrumentation footprints; reducing the level of technical expertise needed for machine operation; and reducing unit and sample processing costs for clinical application.

Commercialization
Despite the formidable challenges there is much excitement around single-cell genomic DNA analysis, particularly in cancer care, and proof-of-principle studies are beginning to emerge. Yet, to date, trials are small scale and the technology still has to meaningfully enter clinical practice. Experts say that while prototypes exist, the technology requires further investment to ensure affordability, manufacturability, and repeatable performance before commercialization is viable.

“The future of microfluidic cell sorting is promising,” writes Shields. “With the large variety of cell sorting devices … microfluidic cell sorting technologies can offer speeds and accuracies in a number of ways that rival current commercial devices in a platform that is more efficient, less cumbersome, and offers a more straightforward standard operating procedure.”

DTET spoke to two early-stage companies about their technologies and next steps to bring single cell sorting technologies to the commercial market.

“We think of it like a sandwich. If the meat is the sequencing, one bread is data analysis and the other bread is sample prep,” Jose Morachis, Ph.D., CEO of NanoCellect Biomedical (San Diego) tells DTET. “New [sample prep] technologies like ours are the exciting next frontier in sequencing.”

NanoCellect’s WolfCell Sorter platform is based upon microfluidic technology licensed from University of California, San Diego. The company believes the portable, bench-top platform addresses some of the current limitations and accessibility of flow cytometry technology. The platform integrates novel detection algorithms with on-chip sorting that uses a piezoacoustic actuator to gently direct cells into collection channels. The technology reduces shear stress on cells, while the disposable, closed chip eliminates dangerous aerosols and potentially brings flow cytometry abilities to more laboratories, the company says.

The integration of cell-sorting and single-cell positioning will allow diverse downstream applications—including kinetic analysis of secreted particles, genomic sequencing, gene expression, and imaging. Morachis says the company’s technology enables advancements in the CTC field, as well as potentially stem cell and CRISPR applications. The cell sorter is currently in beta testing, with a larger release of the $100,000 platform for the research market expected in the summer of 2016.

Liquid biopsies of CTCs are considered the low-hanging fruit of the single-cell analysis market. While evidence of clinical utility is still necessary to further adoption, experts say the application is already showing signs of “maturing.”

Silicon Biosystems (a subsidiary of the pharmaceutical company Menarini Group; Italy), however, has its eye set, not just on the liquid biopsy market, but also on capturing the market for solid tumor samples that are currently “discarded” by molecular labs for insufficient content of tumor cells—currently estimated to be as high as 20 percent to 30 percent of samples.

“We demonstrated we can recover it for NGS analysis even if the sample only has 5 percent tumor cells,” Raimo Tanzi, Silicon Biosystems’ chief commercial officer, tells DTET.

Molecular analysis from formalin-fixed paraffin-embedded (FFPE) samples characterizes all DNA present, including DNA from tumor cells, stromal cells, and infiltrating lymphocytes. Thus, the relative frequency of a given mutation in the heterogeneous mix of DNA molecules is all that can be assessed, while copy number aberration and other genomic instabilities are very difficult to detect, the company says. Its DEPArray system can be used to separate tumor cells from stromal, and other cells in the FFPE section and a “highly pure” collection of tumor cells can be recovered enabling complete genomic analysis.

The company’s DEPArray platform utilizes a semiconductor controlled flow chip where individual cells are coupled to single microelectrodes by dielectrophoretic (DEP) forces. The system combines the ability to manipulate individual cells with high quality image-based cell selection. The company says the technology identifies and sorts individual cells, or pools of cells, with 100 percent purity for detailed molecular analysis. Tanzi says the company will launch pure FFPE tumor analysis as a service at its San Diego-based CLIA lab later in the year.

New Technological Approaches In the last year many academic research groups published studies on novel sorting technologies. Below is a sampling.

• Parallel Microfiltration Method (PMF) is a screening method that utilizes cell squishiness to classify cancer cells. The researchers, from University of California, Los Angeles, say cancer cells are generally two to five times squishier than normal cells, with pliability similar to that of Jell-O. PMF enables simultaneous measurements of cell mechanotype across multiple samples by using uniform air pressure to drive cell suspensions through porous membranes. The relative “deformability” of a cell sample is quantified by the fraction of sample retained above the membrane, with stiffer cells blocking the pores and squishier cells allowing more of the celland- liquid mixture to pass through. The team found that drug-resistant human ovarian cancer cells are softer than their drug-sensitive counterparts, and that more-invasive cancer cells are softer than less-invasive ones. (The technology was described in a study published Dec. 2, 2015 in Scientific Reports.)

• Acoustic Separation Method (“Acoustic Tweezers”) works by using tilted-angle standing surface acoustic waves to apply pressure to a continuous flow of blood. Based on their size and weight, cancerous cells are forced out of the stream into a different channel, where they are collected. The power, intensity, and frequency used to generate the waves in this study are gentle (similar to common ultrasounds), the researchers say, helping to preserve the biocompatibility of the CTCs. The researchers, from Pennsylvania State University, report a throughput rate 20 times higher than previously achieved with similar devices, along with a separation rate of more than 83 percent. The researchers also say they found a way to separate the fluid-containing part of the device from the more expensive ultrasound-producing piezoelectric substrate, making “disposable” acoustic tweezers a cheap possibility. (The technology was described in a study published in April 2015 in the Proceedings of the National Academy of Sciences.)

• Microstructure-Constricted Filtration and Pneumatic Microvalves combine in this integrated microfluidic device to separate cells based on size and deformability. The researchers, from Northwest A&F University in China, say the technology can separate cancer cells from blood samples with more than 90 percent cell recovery and 80 percent purity. In a test of mesenchymal-like cancer cells and epithelium-like cancer cells (with different deformability) the technology demonstrated a “high” selectivity and recovery rate, the authors say. The high viability of the target cells benefits the ability to conduct downstream analysis. The device can also capture and release cells repeatedly, which improves its scalability. (The technology was described in the Aug. 2015 issue of The Analyst.)

• Thermoresponsive NanoVelcro is a postage-stamp–sized chip with nanowires that are 1,000 times thinner than a human hair, the researchers say, and are coated with antibodies that recognize CTCs. When 2 milliliters of blood are run through the chip, the tumor cells stick to the nanowires like Velcro. To separate the cells from the chip without damaging them, the purification system raises and lowers the temperature of the blood sample to capture (at 37 degrees Celsius) and release (at 4 degrees Celsius) circulating tumor cells at their optimal purity. The “mild” changes in temperature, the authors say, allow minimum disruption to the cells’ viability and molecular integrity. The UCLA-based researchers were able to successfully conduct mutational analysis of the purified CTCs from lung cancer patients sorted by this system. (The technology was described in the January 2015 issue of ACS Nano.)

Takeaway: A flurry of recent papers testing new methods and applications for single cell sorting attest to the excitement building around the clinical potential for the technology, including in the areas of CTC liquid biopsies.

Microfluidic cell sorting Technologies
Types of sorting (Based on Cell Preparation)	physical principles (Used For Sorting Mechanism)
Fluorescent Label-Based Sorting - relies on fluorescent probes or stains to identify cells by type.	Electrokinetic Mechanisms Acoustophoresis Optical Manipulations Mechanical Systems
Bead-Based Sorting - depends on particles of a specific material, size, and surface-binding capacity to cap- ture target cells, but can manipulate groups of cells simultaneously and holds promise for isolating tumor cells from unmodified biological fluids	Magnetophoresis Acoustophoresis Electrokinetic Mechanisms
Label-Free Sorting - relies on the physical differences in the properties of cells (size, shape, density, elasticity, polarizability, and magnetic suscepti- bility) and generally requires the least amount of preparation	Acoustophoresis Electrokinetics Magnetophoresis Optics Passive Cell Sorting
For more information see Shields’review“Microfluidic Cell Sorting: A Review of the Advances in the Separation of Cells from Debulking to Rare Cell Isolation”published in Lab on a Chip, Feb. 16, 2015.