Cell Sorting Advances Extend Its Reach

Cell sorting has come a long way since Mack Fulwyler developed the very first cell sorter back in the 1960s. Now, rather than detecting just one fluorescent readout along with forward scatter, cell sorters are able to measure multiple parameters simultaneously—meaning they underpin a growing number of applications. In addition to the established practice of enriching live blood cell subsets, cell sorters are used for stem cell research, cell-line development, monoclonal antibody production, and a broad range of ‘omics techniques, to name but a few. In this article, we explain how cell sorting has become easier, gentler, and more efficient, consequently extending its reach.

Cell sorters are becoming easier to use

According to Brandon Williams, Senior Product Manager at Bio-Rad, a main advantage of newer cell sorters over earlier models is that they offer a greater number of detectors and lasers, allowing researchers to be more targeted. However, this can make cell sorting more challenging, especially for those who have little experience with flow cytometry. “In this respect, one of the greatest cell sorting advances has been the development of instrumentation that is getting as close to approachable as a PCR machine,” he says.

“Although it’s incredibly useful to monitor multiple parameters in parallel, many cell sorting applications require just one or two lasers and a couple of detectors. Bringing these types of [user-friendly] sorters into the lab can help streamline simple sorts by reducing researchers’ dependence on dedicated operators. One of the ways the S3e Cell Sorter  helps make this a smooth transition, for example, is by using a ribbon-based layout that resembles other software such as MS Office suite, so there’s already an element of familiarity when you’re learning to use the system.” 

Cell sorting is getting gentler

A main advantage of conventional cell sorters is that they are high throughput, making them ideal for applications that require large numbers of cells. Yet, a drawback of these systems is that cells may experience sorter induced cellular stress (SICS) due to the high pressure used for running the samples, electrostatic charging, and the frequency of high-speed collisions.

“Microfluidic chip-based sorters are growing in popularity, particularly among researchers who are handling delicate or rare cell types,” reports Jin Akagi, Ph.D., CEO of On-chip Biotechnologies USA. “Several companies offer these instruments, all of which have different features and benefits. Our system, the On-chip Sort, manipulates cells within a microfluidic chip using a method called flow shift cell sorting. This eliminates many of the damaging steps involved in conventional sorting, and thus reduces the overall stress cells experience as they go through the sorter.” A further advantage of the On-chip Sort is that it allows the sheath fluid to be tailored to the cell type for improved viability, for example helping to preserve marine organisms that have been sampled from the ocean. It also enables the collection of large cell clusters, such as cell spheroids and organoids, that are increasingly used as model systems.

Cell sorters are smaller and safer

NanoCellect’s WOLF cell sorter is another option for researchers wishing to perform microfluidic chip-based cell sorting. Lindsey Wolf, Product Manager, explains that the system ensures operator safety by using sterile, disposable cartridges to eliminate the risk of aerosol generation, and by being small enough to fit inside a biosafety cabinet—meaning it has been widely adopted for infectious disease research.

“Single-cell analysis helps researchers to better understand the nature and complexity of different diseases, and promises to unlock more effective therapies,” she says. “But it is vital that end users are protected from exposure to potential biohazards and that samples do not get cross-contaminated—two major issues in conventional cell sorting. Microfluidic technologies have not only reduced the footprint of cell sorters but have also decreased the risk of cross-contamination, translating to safer working practices and more reliable data, respectively.” Chip-based systems extend these benefits even further, making them well-suited to applications where it is critical that the characteristics of sorted cells remain unchanged. Examples include genomics applications, where the high pressure and shear stresses produced by conventional sorting can lead to altered gene expression profiles, and the isolation of cell types such as neutrophils that can become activated during traditional sorting to affect the integrity of downstream assays.

Fast, gentle sorting and isolation of single cells in one step

“In general, cell sorting refers to the enrichment of cellular populations and it is worth noting that microfluidic-based cell sorters typically cannot isolate single cells,” reports Junyu Lin, CEO at Namocell. “Yet, with so many applications requiring individual cells, it’s important that technologies keep pace. To make single cell sorting and isolation faster and easier, our Single Cell Dispenser technology uniquely combines flow cytometry, microfluidics, and liquid dispensing to identify and isolate individual cells in a single step. This preserves the gentleness, sterility, and biosafety of microfluidic sorters, while at the same time provides the high throughput needed for selection of low abundance cells such as circulating tumor cells (that have clinical utility as biomarkers for the management of various cancers) and circulating fetal cells (for non-invasive prenatal testing).”

Other applications benefiting from Namocell’s approach include cell-line development/engineering, where the low sorting pressure (<2 psi) helps improve clonal outgrowth; single-cell genomics studies, where preventing the induction of stress genes is critical to avoid complicating results; and sorting precious cell samples which cannot be sorted with FACS.

Image: One-step cell sorting and isolation. Illustrated detection channels are for the Hana Single Cell Dispenser. Image provided by Namocell.

Future developments

As cell sorting technologies evolve, methods that offer faster, gentler, more intuitive sorts are receiving considerable interest. “At present, it’s really a race to be able to sort cells using more parameters while making instrumentation for simple sorts as easy to use as possible,” comments Williams. Wolf adds that spectral flow cytometry (a technique that reads the emission spectrum of a fluorophore rather than a discrete range of wavelengths) is helping to address the former by providing greater flexibility in panel design. “A major difficulty in high dimensional flow cytometry and cell sorting is finding combinations of fluorophores and antibody conjugates that minimize the need for compensation,” she says, “but spectral flow cytometry has certainly expanded researchers’ choice when it comes to reagent selection.”

Akagi notes that as sample types diversify, researchers may find that conventional approaches to cell sorting are no longer applicable. “The emergence of novel cell sorting technologies has allowed enrichment of rare or sensitive cell types and has been fundamental to new discoveries being made,” he says. “Newer systems provide a wealth of opportunities for researchers who have historically faced challenges arising from the inherent limitations of conventional methods.”

Lastly, Lin notes that modern innovation has made sorting and isolating single cells faster and easier, while also addressing the need to sort a wider range of cell input and cell abundance. “The ability to quickly isolate rare cells such as circulating tumor cells and fetal cells, and to sort precious samples (e.g. clinical samples) that are available in only limited quantities, will facilitate clinical studies and the development of essential therapeutics,” he says.