Overcoming Limitations of Conventional Cell Sorting

Cell sorting allows researchers to isolate rare cell types from a heterogeneous population, increasing the number of cells available for downstream analysis. However, conventional sorting can be a harsh process that not all cell types can go through unharmed. This article discusses a myriad of strategies that were designed to improve on established methods. A recent area of concern is how to avoid SICS, or sorter-induced cellular stress, which entered the flow cyometry lexicon in 2018 (DeLay et al.) and was further investigated by Pfister et al. in 2020 and by Ryan et al. this year.

Variety of methods

Fluorescence-activated cell sorting (FACS) is one of several methods for extracting specific target cells from a heterogeneous sample, with other techniques including density gradient centrifugation, microfluidic cell sorting, buoyancy-activated cell sorting (BACS), and magnetic-activated cell sorting (MACS). Choosing between these approaches depends largely on the required purity, yield, and speed, as well as on cell viability and functionality after extraction. While density gradient centrifugation has limited throughput and specificity, the two most common methods, MACS and FACS, improve on these parameters in different ways. “The main difference between MACS and FACS is that MACS provides bulk separation of cells, whereas conventional FACS is based on single-cell sorting,” explains Dr. Felix Eppler, Global Product Manager for Cell Sorting at Miltenyi Biotec. “MACS consequently has the higher throughput of the two techniques, while FACS utilizes more parameters and, thus, is more specific.”

Limitations of conventional sorting

A drawback of conventional sorting is that, in some cases, it can reportedly induce SICS due to the high pressure, electric charge, and shear forces involved, as well as the lack of temperature control throughout the process. Long sort times can also compromise cellular viability and restrict researchers to isolating just one or two populations. Other limitations are that the fluidics system cannot easily be exchanged, and droplets and aerosols are produced in open space—creating potential safety issues for the operator. 

Strategies to prevent SICS 

Despite its limitations, traditional sorting is still widely employed, with various approaches having been developed to overcome SICS. These include ensuring cells are healthy and viable pre-sorting, controlling the sample temperature, filtering the sheath fluid and storing it at room temperature away from direct sunlight, modifying the nozzle tip size and pressure, and optimizing the pre-sort buffer. Notably, Eppler highlights a recent publication showing that using complete media as the sheath fluid can largely rescue the intracellular markers of SICS. “Although this is an interesting finding, the question remains whether this workaround is a practical solution,” he cautions. “Burning large quantities of complete media during a sorting process may not be a feasible approach.”

Newer sorting technologies improve on conventional sorting

Technologies designed to improve on conventional sorting incorporate a range of features for gentler, safer, more consistent sorting of cellular populations. For example, Thermo Fisher Scientific’s Bigfoot cell sorter uses jet-in-air sorting rather than conventional cuvette-based sorting to help minimize SICS, and additionally mitigates the risk of cellular damage through integrated temperature control. “Another key feature of the Bigfoot is the use of real-time spectral unmixing for fluorophore identification and cell sorting,” notes Angie Goldfain, Senior Manager for Research and Development. “This increases experimental consistency, both between users and between sorts performed on different days. Population identification is further enhanced by dedicated small particle detection for better resolution of scatter signals.”

As an alternative to droplet-based sorting, Miltenyi’s MACSQuant^® Tyto^® integrates microfluidics with micro-electro-mechanical systems (MEMS) to gently perform the entire cell sorting process within a sterile cartridge. “The design of the MACSQuant Tyto  eliminates sample-to-sample carryover or cross-contamination and avoids the production of droplets or aerosols,” says Eppler. “This facilitates translational and even clinical applications—including the recent treatment of a melanoma patient with sorted antigen-specific T cells—and allows for use in a Biosafety Level 3 containment setting.” The MACSQuant Tyto has also contributed to researchers’ understanding of the humoral immune response to SARS-CoV-2 by enabling SARS-CoV-2-specific B cells to be sorted from the blood of convalescent COVID-19 patients.

In recent years, it has become possible to both sort and isolate individual cells gently in a single step. To achieve this, Namocell’s Single Cell Dispensers combine flow cytometry, microfluidics, and liquid-dispensing technologies into a cartridge-based system, whereby changing fluid dynamics are used to sort and dispense the cells into 96- or 384-well plates. In a study published by the National Institutes of Health (NIH) Stem Cell Translation Laboratory in Nature Methods in May 2021, this approach facilitated the discovery of CEPT, a four-part small-molecule cocktail that protects iPSCs from cell stress and improves their viability. This discovery has the potential to accelerate a broad range of applications in stem cell research and regenerative medicine, from genome editing, biobanking, and organoid formation, to cell therapy. “Challenges of conventional single-cell isolation methods include compromised cell viability/functionality, risk of contamination, operational complexities, and the need for large amounts of starting materials,” explains Junyu Lin, Namocell CEO. “Addressing these issues has opened up new opportunities for single cell-based research and discovery.”

Caption. Microfluidic technology enables sequential sorting without compromising cell viability. In this experiment, three different subpopulations were sorted out of one population of peripheral blood mononuclear cells (PBMCs), starting with CD19⁺ B cells for the first sort. The negative fraction was used as the input for a second sort, enriching CD8⁺ cytotoxic T cells. In the third sort, the negative fraction of the second sort was used as input to sort for CD14^–/CD3^–/CD56⁺/CD16⁺ NK cells. All fractions showed viabilities greater than 98%. Image provided by Miltenyi Biotec.

References

1. DeLay, M., Lopez, P., Schiemann, M. Cell sorting for function and viability. CYTO 2018, Prague, Czech Republic.

2. Pfister G, Toor SM, Sasidharan Nair V, Elkord E. An evaluation of sorter induced cell stress (SICS) on peripheral blood mononuclear cells (PBMCs) after different sort conditions - Are your sorted cells getting SICS? J Immunol Methods. 2020 Dec;487:112902. doi: 10.1016/j.jim.2020.112902. Epub 2020 Oct 15. PMID: 33069766.

3. Ryan K, Rose RE, Jones DR, Lopez PA. Sheath fluid impacts the depletion of cellular metabolites in cells afflicted by sorting induced cellular stress (SICS). Cytometry A. 2021 Sep;99(9):921-929. doi: 10.1002/cyto.a.24361. Epub 2021 May 24. PMID: 34031988.