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Cell Microsystems Targets Single-Cell Gene Editing, NGS With Automated Cell Sorting Technology

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NEW YORK (GenomeWeb) – With the recent commercial launch of its array-based AIR system for single-cell sorting, Cell Microsystems aims to tap into the growing markets for gene editing research and single-cell sequencing.

Cell Microsystems also aims to engage the sequencing sample prep market by eventually developing a fully integrated imaging and RNA-Seq library preparation system based on sequencing research at Columbia University, according to company officials.

Founded in 2010 based on technology developed in the lab of University of North Carolina, Chapel Hill researcher Nancy Albritton, Cell Microsystems is attempting to simplify the cell-sorting workflow that researchers use as part of next-generation sequencing, clonal colony propagation, and single-cell genomics.

In 2014, the 10-person, Research Triangle Park-based firm"rebooted" under current CEO Gary Pace and spent the next few years deciding how to best utilize its core technology, called CellRaft, for different research applications.

"From there, we've pursued single-cell genomic analysis, as well as CRISPR editing workflows, enabling single-cell cloning growth in those areas," explained Nick Trotta, the firm's director of product applications and market development.

The CellRaft system is composed of individual "CellRafts" contained in a  "CytoSort" array of 10,000 (200-by-200 micron) or 40,000 (100-by-100 micron) microwells. Researchers feed cells onto the CytoSort array, allowing the cells to settle into the microwells and attach to the CellRaft within each resealable microwell.

Once the cells settle into each well, the researchers pierce the bottom with a small needle that pressures the raft out of the array. Once the raft is released, a magnetic stick picks up the raft and drops it into a standard 96-well plate or PCR tube, which allows the single cell or colony to expand into a larger colony for subsequent genomic analysis. Using the manual system, researchers can produce samples ready for downstream analysis in about three to four hours.

"We can isolate the single cell, grow a small colony on the array, then release the clonal colonies at any time," Trotta explained. "You can collect your sample on day four, day eight, or later," all from the same pool of cells seeded on the CytoSort array.

Cell Microsystem's new AIR system integrates fluorescence microscopy to automate the sample prep process with advanced selection features, speeding up the overall process. According to Trotta, each run requires less than 90 minutes to sort 96 individual cells.

Trotta noted that the CytoSort array is compatible with both adherent and non-adherent cell lines. However, researchers must apply a coating on the array for non-adherent cells such as B cells, T cells, and blood cells. While Cell Microsystems has used primary cells from mouse lineages, the firm hasn't applied any primary human cells at this time.

Through multiple Phase II Small Business Innovation Research grants from the National Institutes of Health, Cell Microsystems has partnered with several US academic institutions — including Columbia University, the University of Virginia, UNC, Washington University in St. Louis, and the Albert Einstein College of Medicine — to further validate its cell sorting system for different research applications.

Albert Einstein genetics professor Jan Vijg explained that his team uses Cell Microsystems' technology to examine somatic mutations using downstream genetic sequencing analysis.

"We needed to look at single cells that had de novo mutations, but we discovered that there were not any good systems currently available to help look at mutations and variants in cells after the amplification step," Vijg said.

Learning about the CellRaft platform from a former Albert Einstein postdoc who saw the tool at a Cold Springs Harbor Single-Cell Analysis conference a few years ago, Vijg realized that it could aid his team in separating single cells into tubes for observation. Since then, Vijg's team has transitioned from manual sample prep to the AIR system earlier this year for faster results.

"The new tool is convenient, as it can scan all of the array at once, and you can select which cell you want to use based on the image, and put it in the tube," Vijg's lab manager Moonsook Lee said, noting that manually isolating the cells was very difficult and inefficient.  

However, Trotta acknowledged that the firm's biggest challenge developing the CytoSort and AIR tools has been ensuring that they remain flexible for the variety of experiments customers want to perform.

"The technology has so much capability that we are still trying to make sure the instrumentation, consumables, and software have all the capabilities our users need," Trotta said. "The challenge with a highly flexible platform technology is ensuring we 'future-proof' the products to accommodate new workflows as they arise."

As an example, Trotta pointed out that several academic labs collaborating with Cell Microsystems on single-cell genomics have also developed an interest in sample prep for downstream gene editing applications, for instance with CRISPR.  The firm is now developing a software platform capable of tracking clonal colony growth, which it plans to release in the "coming weeks."

"[Because] you're editing the genome, there's going to be an impact on physiology and growth rate, but no one's sure how fast their cells are going to grow once they've introduced the mutation," Trotta explained. "Being able to dynamically [select] from a single cell consumable from the same transfection reaction helps users to get a better sense of growth rate and not have to trypsinize an entire plate."

While the AIR system can quickly observe cells that researchers would like to isolate and cultivate in the wells, Vijg noted the software struggles to detect polyploidy, such as diploid and tetraploid liver tissue. However, Vijg said that the firm is currently developing computational software to solve these types of issues.  

Cell Microsystems said that it has exclusively licensed several patent families from UNC, in addition to four previous patents covering the CellRaft technology. The firm is also waiting for patent approval in the US, Europe, and Australia for patent families "jointly owned with UNC."

Cell Microsystems is not only the company aiming to tap into the market for individual cell sorting and analysis.

Fluidigm was one of the earliest companies to tap into the market with its C1 single-cell genomics system, launched several years ago. The C1 uses integrated microfluidic circuits to enable users to prepare single-cell templates for mRNA sequencing, DNA sequencing, epigenetics, or miRNA expression.

More recently, startup NanoCellect Biomedical's WOLF cell sorter and N1 single cell dispenser uses flow cytometry and microfluidics to separate labelled cells into collection channels. The firm's technology requires less than 45 minutes from solution to cartridge.

"Our system is similar to FACS, but we sort cells via imaging, with the cells already on the array, and then we image repeatedly over the course of days to determine not only transfection positivity, but also which cells are developing into good-looking colonies," Trotta said. "The WOLF sorter… addresses the ease of use and shear forces associated with FACS, but [the tool] still does not provide imaging."

According to the Cell Microsystems, the AIR system stands out from other single-cell sorting tools on the market because it offers "a higher degree of detail" when selecting individual cells.

Trotta also claimed that the tool's efficiency is much higher than other cell-sorting workflows.

"On the array, the colony has an 80 to 85 percent chance of survival once you transfer it to the 96-well plate, versus the [average] 10 to 15 percent colony survival rate for FACS" Trotta said. "[Researchers therefore] can selectively choose which colony [to use] without disturbing the neighboring colonies, allowing them to grow through the period where they're most likely to die."  

Vijg also noted that using FACS-based tools for cell sorting is challenging because researchers cannot see what sample is being inserted into the tube for downstream analysis. In addition, he pointed out that the cell sorting tool can also damage the desired cell or colony the researcher wishes to observe for genomic sequencing.

Cell Microsystems also argued that the CytoSort array replicates standard cell culture conditions, avoiding the appearance of "spurious phenotypes" and enabling "the growth of clonal colonies from single cells." The firm said that the AIR system is easy to use and requires "effectively no set-up prior to use."

Commercialization Efforts

Since renegotiating a previous exclusive commercialization license with Qiagen to a non-exclusive agreements in December 2017, Cell Microsystems has been working with other undisclosed firms to validate its sample prep technology.  

According to Trotta, the firm aims to partner with "core facilities" that have a need for several "different types of single cell or single colony isolation, especially facilities that use flow-based sorting for standard and high throughput samples."

Trotta also noted that the firm is also interested in working with pharmaceutical companies, as "there really isn't a good solution for growing small clonal colonies and doing imaging-based tracking of those [cells], so we're seeing interest to solve the bottlenecks," especially in high-throughput pharmaceutical labs.

While Cell Microsystem's tools are currently for research use only, Trotta noted that the firm believes there is enormous potential in coupling imaging and genomic analysis on a single-cell level. As more next-generation sequencing diagnostic tests become available, the firm sees the move toward NGS technology as inevitable and aims to capture a piece of the sample prep market.

Cell Microsystems sells its manual, inverted microscope system for $2,200, the AIR system for $96,000, and CytoSort arrays for $260, sold in packs of five or 10. However, Trotta said that the firm also offers academic institutions a 20 percent discount for the CytoSort packs and a price of $79,000 for the AIR system.

According to Trotta, Cell Microsystems has so far placed five AIR instruments in "a mix of commercial installations and collaborative research programs," and it plans to place an additional five instruments by the end of 2018. Cell Microsystems also aims to launch a few new variations on the CytoSort array later this year. The firm will soon release the CytoSort HR, which Trotta explained will replace the tool's plastic component with a "glass-like" component in order to improve its imaging quality. In addition, the firm expects to launch "HexaQuad" 24-well plates with each well containing individual cell raft microarrays, which Trotta said will "run high-throughput CRISPR applications with potentially 24 different guides."

Cell Microsystems also anticipates commercial launch of a MegaCyte Array — a 96-well plate in which each well will have several hundred thousand CellRafts — in Q1 2019. Trotta explained that the assay will be used by customers who are interested in identifying rare cell types out of mixed populations from a single sample.

According to Trotta, an early version of this technology is currently being used for rare cancer immunotherapy research at a lab run by UNC associate professor Paul Armistead.

"The goal is to identify which T cells are the best tumor killers and to figure out their exact mechanisms, as [we] could clone and expand that further as a cancer immunotherapy therapeutic," Trotta explained.  

Cell Microsystems has also partnered with Columbia professor Peter Sims, who developed a new technology called single-cell optical phenotyping and expression sequencing (SCOPE-Seq), a workflow that enables researchers to image single cells prior to being fully integrated into single-cell RNA-Seq library preparation.

Cell Microsystems aims to apply the SCOPE-Seq workflow as part of its long-term goal to image single cells to characterize unique phenotypes, while also fully integrating NGS sample preparation into a new instrument it calls "AIR-FLOW." The firm expects to commercialize the new device for RUO within the next two to three years.  

If Cell Microsystems eventually plans to commercialize its technology for clinical use, Vijg speculates that the researchers could potentially use it for prenatal diagnostics or cancer detection, including liver and breast cancer. Instead of searching for mutations in specific genes, Vijg envisions examining blood cells directly to see if they could be characterized as having a high number of spontaneous mutations.

"In this case, we'd like to see an assay developed for blood samples, for example, and take a few cells and see how many mutations there are on average," Vijg explained. "The whole idea is to see if people who have more mutations in, say the brain or liver, might be more susceptible to cancer."

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