Flow cytometry and cell sorting are two distinct yet complementary techniques. Both rely on antibodies to detect specific cells within a heterogeneous population, but while flow cytometry measures the proportion of each cell type, cell sorting does more. Based on the cytometry measurements, cell sorters isolate one or more cell populations from the pool. As demand for single-cell technologies continues to grow, flow cytometers and cell sorters are evolving to meet researchers’ needs.
Sorting is a sample-prep starting-point
While people often refer to flow cytometry and FACS (fluorescence-activated cell sorting) interchangeably, FACS specifically refers to the technology associated with cell separation. “FACS is, in principle, the same as flow cytometry, and follows the same immunostaining protocol,” explains Kenta Yamamoto, product manager, flow cytometry at BioLegend. “However, after FACS, the cells of interest are retained for further study rather than being discarded.” Nick Trotta, director, product applications and market development at Cell Microsystems, adds that flow cytometry can be seen as an analytical endpoint, whereas sorting is a starting point for sample preparation.
“There are generally two main reasons for researchers to sort cells,” says Trotta. “For single-cell molecular analysis, cells are usually sorted by phenotype and then lysed to interrogate nucleic acid, protein, or metabolite content. For clonal colony generation, which is common in stem cell laboratories or in generating CRISPR cell lines, investigators isolate an individual cell and expand it to form a population. In both cases, it is crucial that sorted cells are healthy, verifiable as singles, and phenotypically characterized with extremely high accuracy.”
“Another common downstream use for sorted cells is to incorporate them into a functional assay, for example studying the effects of stimulating a particular cell type,” notes Yamamoto. “Alternatively, one can employ magnetic cell separation, such as our MojoSort™ magnetic cell separation system, as a faster, cheaper alternative to FACS. Although this approach simply isolates cells based on whether or not they are bound to the magnetic beads by virtue of a selected marker(s), it is usually less detrimental to cell health. The development of highly specific monoclonal antibodies, including cell-specific clones, is contributing to the growth of magnetic separation technology.”
A releasable antibody technology delivers many benefits
A caveat of cell sorting is that the detection antibodies remain bound to the cells at the end of the process, blocking the epitopes and potentially impacting on cell function. To address this issue, Miltenyi Biotec developed the REAlease® Fluorochrome Technology. “By allowing the removal of any labels from the cells after sorting, REAlease Fluorochrome Technology frees up epitopes for further downstream experiments, opens up the availability of the fluorescence channels for relabeling, and facilitates sequential flow sorting cycles for the isolation of specific cell subsets,” reports Johannes Fleischer, global marketing campaign manager. “By using REAlease antibodies in combination with our MACSQuant® Tyto® Cell Sorter, researchers can experience the ‘dream team’ of cell sorting”.
Miltenyi Biotec’s MACSQuant Tyto improves on conventional instrumentation by employing a gentler separation method. Cells flow from a cartridge into a specialized microchip containing a sorting valve, where they are interrogated by lasers; a magnetic field triggers the valve to open and directs the target cells into a collection chamber. “This method of sorting brings many advantages,” says Fleischer. “By using a mechanical valve, no charge is applied to the cells. The cartridge ensures sterility and allows complete recovery of the sample in the unlikely event of a clog. Furthermore, consecutive sortings can easily be performed from a single sample to boost yields. This is helpful for the isolation of rare cell populations such as tumor-infiltrating lymphocytes (TILs) or when using precious FFPE material.”
Introducing VACS—a new invention to improve sorting rates
Also working to provide better aseptic cell sorting, Cellular Highways was formed in April 2019 as a spin-out of TTP plc. “Our instrumentation sorts cells by a new invention known as vortex actuated cell sorting (VACS),” explains CEO Salman Samson Rogers, Ph.D. “This is a microfluidic technology with the potential to enable scalable sorting and address the need to improve the speed and sterility of current sorters.”
Rogers says that cell sorting needs to be faster to meet the needs of emerging applications such as cell therapy, liquid biopsy, and phenotypic screening, all of which require processing of more cells. Yet he notes that it is challenging to make a cell sorter faster because cells suffer mechanical stress when forced in a single stream through a nozzle or microchannel. “To maintain viability, rates in a single stream are typically limited to 10–20,000 cells/second,” he says. “So to scale up sort rates, it is necessary that the technology is multiplexed to many identical sorters operating in parallel on a chip. Due to the complexity of cell sorter fluidics and the performance of the optics, nobody has done this successfully until now.”
Elaborating on the concept of parallelization, Rogers describes how Cellular Highways recently demonstrated a 16x parallel sorter chip. “Our entry-level Highway 1 instrument relies on one input and two outputs (sorted cells and waste) on a microfluidic chip,” he says. “We’re also developing an instrument we call Highway 16, containing 16 equivalent sorters to Highway 1, all acting in parallel. This is capable of processing around 160,000 cells/second at high viability, high purity, and high yield and delivers approximately a 10-fold speed improvement on current high-throughput sorters.”
A novel cell sorting technology improves viability
Reiterating that maintaining cell health represents a major issue in conventional cell sorting, Trotta states that Cell Microsystems’ CellRaft™ technology overcomes low viability by culturing individual cells in microwells with access to a shared media source. “384-well plates have historically been preferred to 96-well plates for cell collection since they reduce media volumes,” he says. “However, single cells are not capable of conditioning an entire well and therefore exhibit reduced viability and low proliferation rates. Our CytoSort™ array is a high-density microwell array with thousands of individual wells, each containing a raft—the CellRaft—which serves as a releasable culture site. Cells are cultured as singles but are exposed to the secreted factors of cells in neighboring microwells, resulting in significantly improved viability.”
Image: The working principle of the CellRaft™ technology. Image courtesy of Cell Microsystems.
Trotta notes that the CytoSort array is a consumable for Cell Microsystems’ CellRaft AIR™ system, a platform that allows imaging as well as physical release and transfer of the CellRafts to a culture plate. “Cells plated on the CytoSort array remain positioned on the CellRafts while in culture so that single cells can be isolated or expanded into clonal colonies, ” he says. “This approach to sorting allows researchers to benefit from major reductions in time and cost. For example, in some cell-line development workflows the current standard might take up to two months or more, whereas the CellRaft AIR system can achieve the same outcome in just a week. Moreover, because the technology uses imaging as the sorting modality, it is easy for researchers to verify that a single cell was indeed isolated and to ensure phenotypic accuracy.”
As flow cytometers and cell sorters become increasingly powerful, researchers are benefiting from greater accuracy, improved cell viability, and significant time-savings. This positive trend looks set to continue as instrumentation evolves further still.