Single-Cell Sorting—Technologies, Applications, and Advances

This article delves into the various technologies used for single-cell sorting, including some key applications. It also highlights new and upcoming developments in this rapidly evolving field.

Single-cell sorting technologies

It has been 60 years since the first device capable of separating biological cells was created. During that time, single-cell sorting technology has become increasingly powerful, with the development of platforms that include the following:

• Pala™ Cell Sorter and Single Cell Dispenser—Bio-Techne

“The Bio-Techne Pala Cell Sorter and Single Cell Dispenser uses three core technologies—microfluidics, flow cytometry, and liquid dispensing—to enable a fast, gentle, flexible, and simple process,” reports Ryan McComb, Ph.D., Product Manager, Cellular Analysis at Bio-Techne. “Samples are introduced into a disposable, sterile, microfluidic cartridge, which is loaded into the instrument and pressurized with 1.8 PSI. The cells are then directed down a microfluidic channel where they are interrogated with two excitation lasers, enabling up to 11 different fluorescent channel readouts. Following analysis, users can draw gates around cell populations of interest, analogous to standard flow cytometry workflows. Once a target cell is detected, as per the gating scheme, a high-speed valve opens to allow 1 µL of liquid to dispense the cell into a microtiter plate, leaving non-target cells behind.”

A common application for the Pala is in the development of CRISPR-edited stem cells or iPSCs. “With stem cells, the CRISPR editing efficiency can be low, so having multiple fluorescent labels to select the population of interest is very useful,” says McComb. “The Pala lets researchers use multiple fluorescent probes or viability stains to isolate cells expressing or lacking specific markers. Additionally, the gentle sorting pressure ensures stem cells maintain their undifferentiated state and viability, enabling single-cell clones to grow.”

• UP.SIGHT™ Single Cell Dispenser and Imager—CYTENA

The UP.SIGHT is CYTENA’s flagship single-cell dispensing and clone tracking platform, optimized for cell line development (CLD) workflows. According to Pierre-Henri Ferdinand, Ph.D., Head of Product at CYTENA, the system combines microfluidic dispensing with dual imaging (pre-dispense in-nozzle and post-dispense bottom well imaging) to provide robust, regulator-ready double proof of monoclonality. “The UP.SIGHT is especially effective for sensitive cell types, such as iPSCs, due to its gentle dispensing, which preserves cell viability,” he says. “To support CLD campaigns, the UP.SIGHT integrates seamlessly with C.STUDIO, a clone-centric software designed to streamline documentation and reporting of monoclonality, track clonal outgrowth, and compile clone characterization data to allow for creating hit pick lists. Critically, the UP.SIGHT ensures speed, clone recovery, and traceability in bioprocessing environments.”

CYTENA’s solutions are widely used by major pharmaceutical companies developing therapeutic antibodies. “We have compiled multiple case studies demonstrating how our products enable scientists to generate high-quality monoclonal cell lines faster and with solid proof of monoclonality—a requirement for IND filings and downstream manufacturing,” says Ferdinand. “The UP.SIGHT is also being used to solve challenges for iPSCs in allogenic cell therapy workflows. For example, using the UP.SIGHT, Cellestic (a Belgium-based CRO developing CRISPR-edited monoclonal iPSCs) was able to achieve >50% recovery from single iPSCs, while benefiting from convenient clone tracking and effortless documentation of monoclonality."

• MACSQuant^® Tyto^® Cell Sorter—Miltenyi Biotec

The MACSQuant Tyto Cell Sorter employs single-use, disposable cartridges with a patented microchip technology to provide high-speed, fluorescence-based cell sorting. “Fluorescently labeled cells are loaded into the cartridge under a sterile hood, then the cartridge is inserted into the MACSQuant Tyto,” explains Frank Thiel, Ph.D., Global Marketing and Product Management at Miltenyi Biotec. “Here, filtered air gently drives the cells into the microchip, where they are interrogated by three lasers. When a target cell is identified, a magnetic field is applied to the microchip, triggering a sorting valve to open and direct the cell into a positive collection chamber. The gentle nature of the sorting process preserves cell viability and functionality, while automated setup ensures the system is both fast and easy to use.”

Thiel notes that the MACSQuant Tyto is seeing widespread use for GMP-compliant multiparameter cell sorting in cell therapy, which includes supporting clinical trials covering a broad range of applications and cell types. A 2022 publication explains how the MACSQuant Tyto was used to isolate Treg cells from pediatric thymic tissue, with the aim of preventing rejection in heart transplant children. And a 2023 report describes enrichment of antigen-specific T cells using the MACSQuant Tyto during a first-in-human clinical trial for IMA101, an adoptive cell therapy for patients with advanced metastatic cancer.

• Cyto-Mine^® Automated Single-Cell Analysis and Isolation Platform—Sphere Bio

Sphere Bio's Cyto-Mine Automated Single-Cell Analysis and Isolation Platform utilizes proprietary picodroplet microfluidic systems to encapsulate individual cells in stably generated, picoliter volume droplets. “These picodroplets serve as ultra-miniaturized test tubes, allowing large-scale experiments at the single-cell level,” explains Maryam Ahmadi, Ph.D., Director of Science at Sphere Bio. “Following encapsulation, the Cyto-Mine integrates fluorescence resonance energy transfer (FRET)-based microfluidic screening to analyze, sort, image, and dispense viable single cells into microtiter plate wells, enabling a wide variety of applications.”

An illustrative use of Cyto-Mine technology is in the rapid development of high-producing clonal cell lines for monoclonal antibody (mAb) development. “Key features of Cyto-Mine that facilitate this application include its capacity for high-throughput screening, which accelerates the identification of high-producing clones, and its integrated workflow, which further enhances efficiency and reduces manual intervention,” says Ahmadi. “Additionally, encapsulation of individual cells in microfluidic droplets ensures precise analysis and sorting based on each cell's secretion levels. In combination, these capabilities streamline the identification and isolation of clones with superior production capabilities.”

Latest developments

To address ever-changing needs for single-cell sorting, technologies continue to evolve. “We recently introduced our next-generation automated multi-laser single-cell microfluidic system, Cyto-Mine Chroma,” reports Ahmadi. “With four lasers and four detection sources, Cyto-Mine Chroma enhances efficiency in single-cell isolation, improving the selection process for more robust therapeutic development by allowing multiplexing. In the future, the high-throughput screening and multiplexing capabilities of the Cyto-Mine platform can be used for generating large databases to feed AI models. Generating such data in a cost-effective way is critical to accelerate discovery workflows using deep neural network computer models, allowing more accurate predictions and reducing the time and resources required for candidate selection.”

“The next-generation MACSQuant Tyto is set to launch in the summer,” comments Thiel. “It will bring many new features, like an additional laser and more colors, as well as a brand new software with even more automation and an increased performance. We are also releasing a novel cartridge type, which will enable new applications. Importantly, by maintaining the same gentle sorting characteristics as our current MACSQuant Tyto, our latest instrument will preserve cell health and viability that could otherwise impair downstream processes.”

“As therapeutic pipelines grow, there’s increasing pressure to streamline early-stage development without compromising on quality or regulatory readiness,” says Ferdinand. “At CYTENA, we see this as an opportunity to rethink how technologies interact across the CLD workflow. That’s why we’re developing the C.STATION—an off-the-shelf workcell designed specifically for the CLD space. By simplifying CLD teams’ access to automation, the C.STATION will help biopharma teams push the boundaries of what’s possible in cell line development and cell therapies, faster and more cost-effectively.”