The capability to sort single cells of interest and to deeply characterize them has greatly added to our understanding of biology and disease states. Indeed, cell sorting has become an indispensable tool with a broad range of applications. According to the Smithsonian Institution Archives, physicist Mack J. Fulwyler built the first prototype cell sorter in 1965 at the Los Alamos National Laboratory by joining a Coulter volume sensor with the then newly invented ink jet printer.
Leonard Herzenberg of Stanford University was one of the first to clearly see the invention’s benefits. With Fulwyler’s plans and the cooperation of engineer colleagues, two prototypes emerged to sort live cells using fluorescence. The first employed a mercury arc lamp as a light source, and the second used an argon ion laser to detect cells tagged with fluorescent markers. That was enough to interest the company Becton Dickinson (BD), which introduced the first commercial instrument, the FACS (Fluorescence-Activated Cell Sorter) in 1975.
Cell sorters have come a long way since then, incorporating new advances such as spectral lasers and microfluidic-based technologies. Footprints have shrunk while ease-of-use has significantly increased. What was once an elitist tool requiring highly trained operators is now an accessible, albeit still costly, instrument, especially for researchers with access to a core laboratory with cell-sorting capabilities.
A microfluidic-based work horse
Advances in cell sorting allow scientists to precisely isolate rare cell populations in order to study cellular function and characteristics at the single-cell level. “Single-cell analysis has allowed us to understand the complexity of different disease conditions, which has facilitated drug development for targeted therapies,” said Lina Chakrabarti, PhD, associate director R&D at AstraZeneca.
Chakrabarti’s team generates monoclonal CHO cell lines for production of targeted recombinant protein biotherapeutics. Prior to installing the microfluidic-based Bio-Techne Pala™ Cell Sorter and Pala Single-Cell Dispenser the lab used standard cytometric-based cell sorters.
“These big footprint instruments are complex and require specialized training,” emphasized Chakrabarti. “They also have high maintenance and consumable costs while the Pala platform is easy to use, has a small footprint, and an increased efficiency for single cell sorting.”1
Adherence to regulatory requirements, such as providing proof of monoclonality, is an important facet of the group’s work. In addition to its efficiency, the Pala Cell Sorter delivers high proof of monoclonality, noted Chakrabarti, adding that the cells also do not undergo stress. Sorting is fast and gentle due to the exceptionally low pressure.
“We use fluorescence to get proof of single cells after cell sorting,” explained Chakrabarti. “We also look for cell characteristics, e.g., mitochondrial membrane potential. We have found that high mitochondrial membrane potential corresponds to high recombinant protein production.”
The Pala Cell Sorter offers flow cytometric options with different lasers and fluorescence. For detailed work and deeper dives into cellular features and rare populations a standard flow cytometric-based cell sorter is used. For example, other groups at AstraZeneca work with heterogeneous CAR T cells and require more laser options and parameters. “But these add-ons are not required for what we do,” Chakrabarti said. “For our applications, the Pala Cell Sorter is a workhorse, and the capabilities are sufficient.”
Conventional cell sorters can be intimidating and in the past only Chakrabarti was able to perform all of the sorts. Now, the user-friendly Pala Cell Sorter is available to all the scientists without specialized training.
“In the next generation the addition of imaging, even though it would add additional time to processing, would reduce the time of our current imaging step,” Chakrabarti suggested. “And, automation would be beneficial to reduce manual plate handling.”
Simplicity and versatility
Alfonso Blanco, PhD, director of the Flow Cytometry Core Technologies at UCD, remembers when cell sorters were 1-laser, 3-color instruments. Today, laser capability and the number of detectors, fluorochromes, and antibodies have expanded along with software capabilities, biohazard protection, and self-containment.
“At first, you could only sort one population and now instruments exist that can sort up to six,” Blanco said. “Flow cytometry is moving along with supportive technologies to offer more capabilities to fine-tune our needs. Company approaches vary, which provide choices.”
The Core has the Beckman Coulter® CytoFLEX SRT. “I like simplicity and applications,” said Blanco. “The small footprint CytoFLEX SRT is very flexible, sensitive, and powerful, yet easy to use. It has the same software as the other CytoFLEX analyzers, allowing transition from work on an analyzer to a sorter even with little flow cytometry experience.”
Automated processes provide independent sorting access even to student users after some brief training. As students become more confident they begin to plan more complex experiments. “Users can set up the CytoFLEX SRT themselves with 100% precision, which is important since I am the only staff member at the Core. This makes a huge difference in terms of working towards 24/7 availability, and also from the application perspective.” Blanco emphasized that it is still important to assist users with data analysis, template, and panel design.
The Core’s 20-year-old battle horse, the BD FACSAria™, remains in use mainly to address double bookings, breakdowns, or users with historical experiments that do not want to change experimental conditions. However, it is more complicated to use and less sensitive than the newer instrument.
For almost a decade, Blanco has been involved in sorting extracellular vesicles (EVs). He continues to explore this area using an AcouTrap, which he plans to combine with the CytoFLEX SRT to address the excess volume versus EV quantity dilemma.
The EV scenario highlights the need for high-precision sorting of large quantities of nanoparticles. Potential deterministic lateral displacement (DLD) systems particularly intrigue Blanco as they can separate based on size, morphology, and deformation. The flow sorter for marine biology and microbial oceanography, MarLux, developed by Ger van den Engh, is also interesting as the angle of the lasers can be adjusted for multiple interrogation points as well as the angle of the different scatters.
Game-changing spectral performance
Today, multiparametric cell sorting facilitates research, allowing the isolation of rare cells for further downstream characterization. “In particular, we want to understand a tumor’s progression to a neoplastic stage,” said Thibault Andrieu, Translational and Cytometry Core manager at the Cancer Research Centre of Lyon (CRCL). One of the innovative technologies the core uses is the BD FACSDiscover™ S8.
“We were the first to institute this technology in France. It combines spectral analysis for deep multiparametric phenotyping with real-time imaging,” explained Andrieu. “We can subcategorize, and sort rare populations based on the internal localization of markers within the cells all with one instrument. The spectral performance is a game changer allowing us to focus on complex multiparametric applications.”
Today, the core facility uses the FACSDiscover S8 for applications ranging from simple GFP sorting to complex 30–35 parameter sorts combined, for instance, with a colocation assay for two markers. The previous gold standard, the BD FACSAria, has been used for comparison work for the last year with reproducible results with the new instrument. All work has now been migrated to the new system.
[Translational and Cytometry Core at the Cancer Research Centre of Lyon (CRCL)]
With efficient fixed alignment of the lasers coming from the BD FACSAria, the complex setup is now easily managed on the BD FACSDiscover S8 as well as the physical sorting; it is a new step in managing complex experiments. The transition is straightforward for operators who understand the sorting process.
The Core’s work can often be complex. In these cases, trial runs are performed to test conditions for selection of the key populations. “If you need to pinpoint the localization of the fluorescence and sort specific subpopulations we prefer to preset the experimental conditions due to their complexity,” said Andrieu.
“For these complex studies, consistency of the instrument performance is critical because we apply complex phenotyping to tumor samples from different patients across a long period of time,” he said. “The settings remain stable over time, and we can attribute differences to the sample and not due to drift of the machine.”
A major advance that would aid Andrieu’s work is the development of clustering algorithms to automatically identify specific populations for sorting purposes. The second step would be to combine this capability with AI for machine learning in order to automatically tag these populations.
Reference
- Chakrabarti L et al. Simplifying stable CHO cell line generation with high probability of monoclonality by using microfluidic dispensing as an alternative to fluorescence activated cell sorting. Biotechnol Prog. 2024 May-Jun;40(3):e3441. doi: 10.1002/btpr.3441.
