Dr. James McCracken, senior commercial product manager at Beckman Coulter Life Sciences, discusses how cell sorting is a popular and growing use of flow cytometry – and one that many researchers rely on specialists to perform.
The risk of losing precious cells and samples to user error or low sort performance keeps many scientists at arm’s length from this useful technology.
As a result, cell-sorting technologies have traditionally resulted in high levels of impurities. Earlier generations of cell sorters often caused target cells to die or get attached to unwanted cells and discarded. Many cells that survived became less viable during the sorting process. These impurities made experiments inefficient and costly.
New sorting technology is helping to solve those problems. In fact, advances in cell sorting are making it possible to perform complex experiments that were impossible before.
Expanding cell-sorting capabilities
New cell sorting technologies improve upon traditional systems in several ways, the first of which is by increasing the signal-to-noise ratio. This is achieved by boosting the level of light scatter and fluorescence sensitivity. New designs for light collection at cell interrogation allow for less light loss and therefore more light delivered to the detectors, even when using lower power and less expensive lasers to interrogate. In the same vein, newer detectors such as avalanche photodiodes, silicon photodiodes and GaAs photomultiplier tubes have become integrated into sorter designs to more efficiently amplify light signals into electrical signals.
The increased scatter sensitivity, as well as better staining and sample preparation workflows support research in bacteria, viruses, extracellular vesicles and other very small biological particles, because the increased sensitivity has allowed some manufacturers to separate out standard polystyrene particles as small as 100 nanometers in diameter. Software tools such as FCMPASSTM when used in conjunction with standard particles can support calibration and sizing of nanoscale particles for sorting.
Advances in cell sorting have also improved researchers’ ability to visualise and sort rare events. Modernisation of cell sorter electronics allows for cells to flow through at higher event rates, without overburdening the electronics and missing desired events due to lack of signal processing while dealing with previous events. This allows users to run at speeds that keep active sorting time shorter without missing these events. Less time in the sorter is also important to allow sensitive cells to be returned to optimal conditions more quickly.
Many sorters now offer index sorting when sorting into multiwell plate formats (typically 96 or 384 wells.) Index sorting allows for the separation of data for just the cells placed in wells. This information can be valuable for troubleshooting or when looking for characteristics of cells that might not have been a part of the sort criteria, but are found later to have an impact on cell growth, plate efficiency, or other desirable outcomes.
When cells are sensitive enough to the higher pressures of droplet-based sorting, or when experimental conditions call for absolute sterility, many investigators would be advised to consider microfluidic sorters. While typically lower in throughput in terms of event rate, these sorters typically use single-use fluidics that are sterile, and operate at very low pressures.
Increased access to and adoption of cell sorting is also driven by the design of software that allows the user to set up the hardware to run stably for the entire time of the sort. In the past, most cell sorters were droplet-based and finding the frequency and amplitude to allow for stable droplet formation required a good deal of judgement and experience. More modern software has made this more accessible by the use of wizards driven by software to scan and find the proper conditions. Advanced monitoring tools are also available to maintain stable droplets over time, and even respond to potential problems by pausing the sort and in some cases attempting a corrective action. In other cases, alerts to the user can allow for guided troubleshooting.
Enhanced cell sorting in action
Scientists at Beth Israel Deaconess Medical Center in Boston, Massachusetts, are using advanced sorting technology to study whether CD150-high regulatory T-cells can protect blood stem cells from oxidative stress. The high signal-to-noise ratio has helped enhance signals from rare events. The researchers’ ability to sort cells without use of a UV laser helps ensure they’re sorting on live cells. Their findings could help improve the development of CAR-T cell treatments for cancer.
“We’re getting better results. We’re getting a clearer picture, simply because the signal-to-noise ratio is better, giving us a more enhanced signal,” said John Tigges, technical director of flow cytometry science at the Beth Israel Deaconess Medical Center’s Center for Extracellular Vesicle Research, in a webinar.
Another group at Beth Israel Deaconess with Research Scientist Cecilia Cavazzoni is examining the immune response in viral resistance. They’re sorting B cells after SARS-CoV-2 spike vaccination and researching their ability to accumulate mutations and their interaction with follicular T helper cells.
Meanwhile, a neurology group at Beth Israel Deaconess is studying specific types of T cells that have been found to cause cytotoxic reactions in the brains of people with autism. They are using CRISPR-Cas9 gene editing to test whether changing certain genes in the cells could modulate the cytotoxic effect. At first, the team was struggling with high rates of cell death in the sorting process. So the researchers switched to a 100-micron nozzle with a sheath pressure (PSI) of 15. “The viability was remarkably better,” speeding up the research, Tigges said.
These are just a few examples of the benefits that new cell-sorting systems are bringing to researchers in several therapeutic areas. Improvements in light scatter and fluorescence sensitivity, usability and data tools are offering more opportunities for scientists to separate out specialised cells that were difficult to visualise before. Improving sorting workflow and hardware further should be focused on the overall research goals of yielding more viable, healthy cells to power a wide range of medical advances and directions of inquiry.
