Traditionally, cell sorting has been used for developing cell-based assays, such as those involved in evaluating potential drug candidates. However, cell sorting has been harnessed for many other applications in recent years, driven largely by the emergence of novel sorting technologies. This article provides a quick recap of established methods for sorting cells before taking a closer look at some of the newer alternatives being adopted by researchers.
The use of cell sorting technologies has exploded
According to Brandon H. McNaughton, Ph.D., Chief Executive Officer, and Co-founder of Akadeum, the scientific community is at a unique moment in history where there is a convergence of technology advancement and the utilization of the immune system to study, diagnose, and even treat disease. “Because of this, the use of cell sorting technologies has exploded, roughly doubling over the last five years,” he reports. “Importantly, cell sorting has created access to specific cell types—like B cells and T cells that are taken directly from tissue or blood—which is critical for studying fundamental biology, producing antibodies, and performing single-cell sequencing, as well as for many other applications that are foundational to improving human health.”
Mandana Farhadi, Senior Product Manager for the WOLF Cell Sorter at NanoCellect, agrees, adding that novel research fields such as immune-oncology and cell therapy have heightened demand for sorted cells. “To accommodate researchers’ changing requirements, cell sorters have had to evolve,” she says. “For example, the emergence of multi-omics methods has put an emphasis on the importance of sample preparation. Newer instruments coming on to the market are designed to facilitate modern workflows where traditional cell sorting methods might have limitations.” Ultimately, being able to reliably purify viable cell populations for targeted research or development goals aids the production of effective treatments for conditions spanning rare and infectious diseases through to neurodegeneration and cancer.
Established approaches remain popular
Established cell sorting technologies, including magnetic-activated cell sorting (MACS), remain popular among researchers. Angie Goldfain, Senior Product Manager at Thermo Fisher Scientific, comments that, during conventional cell sorting, cells are stained with fluorophore-labeled antibodies and directed in single file past one or more lasers at an interrogation point before being encapsulated in liquid droplets. “By applying an electric charge to each droplet based on the detected signals, conventional cell sorting allows different cell types to be deflected into tubes or micro-titer plates for collection,” she says. MACS instead uses antibodies conjugated to magnetic beads for labeling cellular markers, enabling specific cell types to be isolated from solution.
Despite their widespread use, these long-standing and highly popular methods are recognized to present certain challenges. “Conventional cell sorters operate at high pressure that could cause cellular damage, resulting in loss-of-function, decreased viability, or reduced clonal outgrowth after sorting,” cautions Farhadi. “While MACS avoids the use of pressurized fluidics, it is limited by the selection of only one marker at a time—and necessitates countless rounds of washing and centrifugation—meaning it is not an effective method to select cells with multiple markers or perform multi-population sorting.” McNaughton points out that, in addition, both technologies can struggle to effectively sort samples containing high number of cells or large fluidic volumes, which is increasingly necessary for applications like cell therapy.
Novel cell sorting technologies address common problems
Several approaches have been developed to address the limitations of existing cell sorting technologies. These promise benefits including gentler, faster, safer sorting, as well as the capacity to detect more markers per sample compared to using conventional techniques.
Buoyancy Activated Cell Sorting (BACS™)
Buoyancy activated cell sorting, also known as BACS, is a method that uses low-density particles (microbubbles) for flotation-based cell separation. McNaughton explains that by functionalizing the microbubbles with antibodies for cell surface markers, BACS provides an incredibly gentle means of cell isolation. “Once attached to the target cells, the microbubbles float to the surface while the other cells drop into a pellet,” he says. “Alternatively, BACS can be used for negative selection, where the desired cells precipitate. In either case, because BACS relies on buoyancy, there are no limits on sample size and no requirement to purchase dedicated equipment.” BACS was recently cited in Cell Reports for purifying murine B cells, which were subsequently used to reveal a role for the orphan nuclear receptor NUR77 during early humoral immune responses.
BACS technology. Microbubbles capture target cells within a sample, which then float to the surface for removal. Image provided by Akadeum.
Microfluidic-based cell sorting
Rather than using high pressure fluidics for sorting cells, NanoCellect’s WOLF cell sorter is centered on microfluidic cartridges. Farhadi notes that this provides gentle sorting in a sterile environment, with no risk of aerosol generation or sample carryover, and also eliminates the need to perform time-consuming daily maintenance. “The WOLF cell sorter is a benchtop platform that is suitable for a broad range of applications,” she says. “For example, cells can be isolated for genomics studies without experiencing shear stress and associated changes in gene expression, or cell lines developed from fragile cell types such as induced pluripotent stem cells. The WOLF also fits easily inside a biosafety cabinet, making it ideal for infectious disease research.” Published uses of the WOLF include modeling of human neuronal cell biology, where preserving the viability of human induced pluripotent stem cells before differentiating them into neurons is essential, and determining how diverse cell populations preferentially acquire distinct metabolites within the tumor microenvironment.
Spectral cell sorting
Spectral cell sorting follows the same basic principles as conventional cell sorting, but differs in how the fluorescent signals are detected. Instead of using individual detectors that each measure a wavelength range corresponding to the emission maxima of a given fluorophore, spectral flow cytometry measures the full emission spectrum of every fluorophore across a detector array, allowing fluorophores with similar emission maxima to be combined in the same panel. “The Invitrogen Bigfoot Spectral Cell Sorter can be configured with up to 9 lasers and 60 detectors,” reports Goldfain. “It also comes with custom-designed Field Programmable Gate Array electronics for real-time spectral unmixing or compensation. Other features that support modern-day cell sorting applications include an integrated biosafety enclosure and aerosol management system, along with multi-way tube and micro-titer plate sorting capabilities, which make the Bigfoot up to 10 times faster than traditional cell sorters.”
Whatever your chosen application, there is bound to be a cell sorting technology available that meets your needs. And, if you have specific questions, manufacturers are always happy to offer guidance.