Optimizing Cell Sorting

Cell sorting technologies are constantly evolving to meet researchers’ changing needs. Here, we look at some of the new capabilities available and the problems they have solved, as well as the opportunities they have ushered in.

Over 50 years of innovation

It has been over 50 years since the first commercial cell sorter  was introduced. Based on fluorescence-activated cell sorting, it exploited the cell-permeant esterase substrate fluorescein diacetate (FDA) for isolating mouse spleen cells, but the technology has since become more powerful with the development of fluorophore-labeled antibody reagents and advanced instrumentation. “Some of the most prominent cell sorting systems on the market today boast 4–6 wells for sorting and can simultaneously detect upwards of 40 fluorescent markers,” reports Mahyar Salek, Ph.D., Co-Founder and CTO of Deepcell. With sorted cells now being used for applications spanning basic research through to clinical diagnostics and therapeutics, it is essential that the technology continues evolving.

Common problems

Cell sorting challenges are centered on panel design, maintaining cellular viability, and avoiding contamination. “The design of fluorescent probe panels can become extremely complex when the goal is high-dimensional analysis,” says Salek. “Compatible probe combinations must be chosen while considering the spectral properties of the dyes, the densities of different antigens, and the relationship between markers. And if you happen to be researching cell properties that don’t have good markers, it becomes increasingly difficult to efficiently characterize and capture populations of interest. In addition, labeling perturbs the cells, limiting their utility for downstream applications.”

Dr. Xin Liu, Director of Technical Support at Sphere Fluidics, suggests that further problems for experimental design can be encountered when there is a need to detect secreted molecules or monitor cell-cell interactions. “Secreted molecules such as cytokines must usually be trapped inside the cells with a protein transport inhibitor such as Brefeldin A or Monensin, which can impact results,” he says. “Measuring cell-cell interactions requires multiple cells to be analyzed and sorted together, which cannot be accomplished using conventional techniques.”

According to Olivier Déry, Director of Marketing at NanoCellect, pressure over time and limited sheath fluid options when using conventional cell sorting systems can compromise cellular viability. “With traditional electrostatic or droplet-based cell sorters, the cells enter the flow cell at a very high pressure, typically 20 to 70 psi, which then rapidly drops as they exit through the nozzle opening,” he says. “This causes decompression shock and shear stress, leading to damaged cells with poor viability, as well as risks altering phenotypic attributes such as proliferation, activation status, morphology, metabolism, and even gene expression. Several cell sorting applications are therefore impacted by these limitations. For example, scientists will observe poor clonal outgrowth for cell line development and CRISPR gene editing workflows or suboptimal cell populations separation in downstream genomics and transcriptomics. These issues can be exacerbated by the requirement to use phosphate buffered saline (PBS) as the sheath fluid, which may not be best for all cell types.”

Contamination is another concern due to the open design of many traditional cell sorting instruments. Not only is the sterilization process for traditional sorters arduous, but it does not guarantee no sample-to-sample crossover or potential exposure to biohazardous aerosols. “With demand for cell sorting technology continuing to grow, driven in part by the increased use of cell therapeutics and regenerative medicine, addressing all of these challenges remains of paramount importance,” comments Chuck Na, Manager of Innovation at ATCC. “When it comes to therapeutic uses especially, the fidelity of the purification and separation method is critical.”

Innovative solutions

Various approaches are being used to address the problems just described, including the following:

Label-free sorting

Deepcell’s REM-I platform combines high-resolution imaging of single cells and label-free sorting with AI in one platform. “A defining feature of the REM-I platform is its artificial intelligence model, the Human Foundation Model,” explains Salek. “This extracts visual features from a large and diverse set of cell images, without prior knowledge of specific cell types, cell preparations or other application-specific markers, and creates massive morphology-based datasets using deep learning and computer vision to quantify over 100 different morphological features of single cells. Using the REM-I platform, researchers can review both individual cell images and groupings of cells, and can sort morphologically desired cells into up to six wells for additional downstream analysis.” To date, the REM-I platform has been used for applications including early and non-invasive detection of organ transplant rejection, enriching for cancer cells in malignant fluid, and distinguishing subtypes of lung carcinomas.

Picodroplet technology

Sphere Fluidics’ cell sorting technology uses picodroplets, miniaturized water-in-oil droplets that are made and manipulated using microfluidics. “Manipulation includes cell encapsulation, incubation, and splitting, as well as reagent injection, sorting, and dispensing, meaning that each picodroplet essentially serves as an ultra-miniaturized test tube” notes Liu. “Because each droplet contains either one or multiple cells, together with any secreted molecules, our technology allows for sorting based on cell-cell interaction, molecular secretion, and many other novel schemes, in a high-throughput manner.” Picodroplet-based microfluidic technology has enabled the discovery and development of antibody therapies by directly measuring the antibodies secreted by single cells and is also being used for cell-cell interaction studies and synthetic biology applications.

Microfluidic cartridge-based sorting

NanoCellect’s WOLF systems are designed for gentle, sterile sorting under optimized conditions. “With the WOLF systems, the change in psi is virtually non-existent, remaining below 2 throughout the sorting process to better preserve the sorted sample,” says Déry. “Sample quality is further enhanced by the use of disposable microfluidic cartridges, which can accommodate different types of sheath fluid to provide optimized conditions and have a fully contained fluidics path for completely sterile and safe aerosol-free sorting. These features of the WOLF systems, plus their compact size, are enabling novel applications. For example, one of our customers recently took a WOLF cell sorter to Antarctica and used it for researching bacteria in sea water samples—something that, to our knowledge, has never been done before.”

Standardizing cell separation

Fang Tian, Ph.D., Director, Biological Content at ATCC, highlights ongoing efforts toward improving refinement and specificity in cell sorting instrumentation and processes. “ATCC is actively involved in standardizing cell separation and provides authenticated materials to the National Institute of Standards and Technology (NIST) for use as standards in cell counting and separation,” she says. “In this way, we have a central role in enabling the effectiveness of advancing technologies, as well as serving to unite the global research community such that improved, more uniform methods for cell separation become available.”

Future perspectives

Cell sorting technologies continue to evolve at pace in response to researchers’ changing demands. “The trend is toward translational research—to use advancements in cell-sorting technology for therapeutic and diagnostic purposes through a better understanding of biology,” says Na. “Additionally, the speed of development in areas including data acquisition, high content imaging, and imaging analysis, plus the requisite decrease in cost of infrastructure, is allowing researchers to move away from traditional forms of cell sorting. Ultimately, this remains a vibrant field of research that is rapidly advancing as the scientific and engineering communities collaborate to “build a better mousetrap.”