Capable of boasting high-throughput, accurate, multiparameter and highly reproducible results, Urmi Roy, Global Product Manager Miltenyi Biotec outlines the benefits of flow cytometry offering case study examples for its use.
Increasing numbers of drug discovery programmes are targeting cellular mechanism and phenotype as a promising avenue for new therapies. The clinical motivation behind this is clear: therapeutic candidates that perturb specific cell types could revolutionise how we intervene in disease progression. Accordingly, we are witnessing the greatest growth in so-called biologics as a class of drugs today.1,2 But understanding the intricacies of cellular biology requires a far greater investiture in cell-based assays than has historically been the case, and with that rising focus, there comes a rising demand to master a huge variety of assays. Mastery in this sense means not just specialisation in a large number of subtly different techniques, but also the ability to make them both rapid and routine, since – after safety – the speed at which effective new drugs reach the bedside is arguably the most critical factor in in successful drug development schemes.
Flow cytometry is a powerful tool to analyse phenotype and cellular assay end points, as well as to enrich and sort specific cells of interest, so it is little surprise that we find the technique used at several different stages of the drug discovery pipeline, including target identification and validation, hit identification, lead and candidate selection, and safety studies.3 Compared with other methods of extracting such information, flow cytometry’s ability to provide multiparameter, high content analysis, single cell resolution, sample multiplexing capabilities, and analysis of large numbers of cells in relatively short period of time makes it an obvious choice for the study of biologics in drug discovery settings. Compelling as those reasons may be, however, it does not make flow cytometry a tool without significant drawbacks – at least as it is traditionally undertaken.
Flow cytometers utilise a sophisticated array of lasers, optics, fluidics, and electronic detectors to measure light scatter and/or fluorescence emission from cells and other particles, for example beads. These bodies are run sequentially and individually through a laser to quantitate physical properties, including size, granularity, and the numbers of target proteins. Choosing and setting these setup parameters, not to mention selecting the most appropriate antibody-dye conjugates and other reagents, as well as later removing them without damaging the target biologics for downstream use, requires significant training, experience, and above all patience. Factor into that the almost arbitrary-seeming way in which reproducible results flitter in and out of the frame across time, instrument, operator, and facility location, and flow cytometric analyses can sometimes seem more art than science. And this is hardly where we want potentially life-saving science to be!
Happily, a silent revolution of sorts in flow cytometry has been underway for some time now. Gone is the once-assumed reliance on single-tube sampling technology with its adherent and frankly unreasonable requirements for manual involvement in sample acquisition. Instead, newer instruments allow plate-based sampling with varying degrees of automation, but all of them speeding up the assay process dramatically. In some cases, additional effort has gone into further automating the technology behind experimental set up and data analysis to make frequently run assays almost completely hands-off, whilst the newest flow cytometers on the market can now easily be integrated into external robotic platforms that allow many typical assays to run with the automation and efficiency of an industrial factory setting.
Processing at high throughput
These advancements in automation undoubtedly hold the potential to significantly increase both the utility and the practicality of flow cytometry applications in drug development on a purely pragmatic basis. But another improvement in the technology that goes hand in hand with this is speed. Drug discovery assays that screen for biomarker, target, or identification of hits from literally thousands of compounds must go through extensive screening processes. Traditional flow cytometers could never the method of choice because of their limited throughput: In a process that already takes years from conception to fruition, further delays are completely out of the question. Yet today, even the early stages of drug development are seeing an increasing requirement for phenotypic screening and multiparameter functional and safety assessments, leaving many in the industry feeling stuck between a rock and a hard place with respect to flow cytometry technology. How refreshing, therefore, to learn that there are several options currently available that are capable of analysing all samples in a 384-well plate in less than an hour! Such an increase in throughput, when added to its multiplexing abilities, means that flow cytometry – far from being a timely burden in the drug development cycle – actually now has the potential to shave months off the schedule.
The only issue then becomes compensating for the sheer number of cells acquired, and faster washing between samples: The faster cells are acquired, the higher the chance that some will be passed over for interrogation (particularly important if the cells of interest are rare); further, increased speed means shorter instrument washing times between samples, and therefore a greater likelihood of carry-over from one sample to the other. A careful balance must therefore be achieved to maintain data quality at statistically viable speeds while simultaneously keeping sample-to-sample carryover rates to a minimum. The data below shows how this can be achieved, in this instance using a MACSQuant Analyser from Miltenyi Biotec to apply high-throughput flow cytometry to phenotype mesenchymal stem cell properties grown in different culture conditions.4
To help identify and characterise off-target effects at the level of the single cell, toxicology studies regularly employ both in vitro and ex vivo flow cytometry methods .5 Flow cytometry is also widely used during clinical testing in the assessment of safety, for example in anti-drug antibody testing, and also in pharmacokinetic assessments when attempting to monitor the plasma levels of protein- and peptide-based therapeutics.6 Beginning with simple cytotoxicity assessment and ranging up to complex immunotoxicity assays, flow cytometry finds a range of uses in the field of toxicity, and the increased interest that is developing in mechanistic insight together with toxicity potential information is only encouraging researchers’ further employment in toxicological studies.
In one such example, Litron Laboratories used a MACSQuant Analyser to apply flow cytometric in place of microscopic analysis in an in vitromicronucleus test. This test is widely used in drug development settings to assess cytogenetic damage in mammalian cells upon trigger. Traditionally employed microscopic analyses lack high-throughput capabilities as a screening tool, whilst Litron Laboratories found its in vitroMicroFlow Kit in combination with the MACSQuant Analyzer 10 provided a fast, standardised, and automated flow cytometry–based workflow that easily overcame those limitations, providing high-content information that was both reproducible and reliable (see figure below).
An added benefit of this setup was that 21 CFR part 11-compliance to maintain electronic records could easily be assured. Many toxicity assays at the preclinical stage require highly regulated GLP conditions, making this no small consideration. Furthermore, since assays often move from one department to another, the ability to transfer settings from one instrument to multiple other instruments makes life a lot easier, and results considerably more reproducible.
Perhaps the biggest impact flow cytometry has had in any field of research since the invention of the technology has been the way it enables complex multiparameter analysis. In drug discovery, this naturally suggests immediate use in phenotyping; However, the technology can also be used in a variety of different applications to greatly influence efficiency, timelines, reproducibility and overall effectiveness. Examples include analysis of microbes, cellular phosphorylation, calcium flux, protein-protein interactions, receptor-ligand interaction assays, and soluble cytokine analysis to name but a few high-impact and easily accessible opportunities to streamline drug development workflows. Such a diverse set of potential applications makes flow cytometry an incredibly versatile tool for almost any drug discovery programme. Moreover, the non-invasive but simultaneously sensitive and quantitative nature of flow-cytometric interrogations make them an excellent choice for delicate operations, for example analysing FRET-based target screening assays.
FRET assays have found extensive use in high-throughput screening as one of the few non-invasive, radiation-free techniques for studying protein interactions within intact cells in real time. The FRET principle is based upon the transfer of energy from an excited donor fluorophore to a nearby acceptor fluorophore, which results in enhanced fluorescence emission by the acceptor.7 This phenomenon can be easily employed to monitor fluorescently labelled protein-protein interactions. In this example, an automated flow cytometry‒based FRET assay8 was performed for high-throughput screening of molecules which have a potential inhibitory effect on programmed cell death protein-1 (PD-1). PD-1 is an immune checkpoint expressed in T cells and pro B cells, with its signaling resulting in inhibition of T cell proliferation, survival, and effector functions. It requires binding to its specific ligand receptor PD-L1 or PD-L2. The former is expressed in several cancers, and therefore drugs targeting the PD-1 signaling pathway are commonly used in cancer immunotherapy.9 PD-1/PD-L1 complex exerts its effect by forming a microcluster in the immunological synapse together with TCR/CD3, and can thus be detected by assays that measure protein-protein interactions. The figure below shows how a flow cytometer was utilized to screen compounds targeting PD1 signaling.
Revolutionising cell sorting
Cell sorting is another important application of flow cytometry. Throughout the process of drug development, cell sorting is a recurring requirement , for example, in isolating antibody-producing B cells, sorting of infected from healthy cells, specific cell subtype sorting for further downstream analyses and applications, and many more. In 2020, this use case has become far more prominent, owing to the emergence and wildfire spread of the COVID-19 pandemic, which has naturally raised concern and interest among scientists worldwide in developing therapies for and preventative measures against such infectious diseases. In such cases, the drug development journey involves a significant number of extra precautions due to the potential risk to scientists and instrument operators handling infectious materials. The traditional cell sorting process is one that is droplet based, and that has shown propensity to generate aerosols, and therefore increased risks of infection. Using such a cell sorter safely would require it to be located in a BSL3-designated room, with all the attendant inconveniences and costs this entails.. Today, though, new technologies combined with flow cytometry principles allows sorting of cells in an aerosol-free and sterile environment, eliminating this requirement altogether and allowing scientists to get to grips with high-risk assays and projects faster.10,11
One such example is the Infectious Disease Unit Research Department at Aarhus University Hospital in Skejby, Denmark; Until recently focused on HIV research, it is now was able to quickly retarget its efforts exclusively towards research into COVID-19. The team there uses plasma from recovered COVID-19 patients, incubates it with the virus, and measures the levels of neutralisation. Good neutralisers are tested against 2002’s SARS virus, and then B cells that can affect both viruses are extracted from patients in order to harvest antibodies that can be used in treatment. They are also using its spike protein coupled to a fluorophore as the bait to mark the B cells that express and produce SARS-CoV-2–specific antibodies.
Although there’s no evidence to date that the virus is still present in recovered patients, the critical step is in sorting their B cells, and no chances are taken, making droplet sorters of any kind completely unsuitable. Instead, the team uses a MACSQuant Tyto Cell Sorter and associated cartridge to make it safe to move samples around to BSL2 or even BSL1 rooms because it´s a fully closed system. It’s also significantly automated, meaning getting hands-on with the instrument requires no in-depth training or highly trained technicians.
The drug discovery process is a long and multi-faceted one, including many different workflows, researchers, technicians, instruments, laboratories, and clinics. Clearly, there is no silver bullet that cuts the timelines in half or more. That said, it is always worth keeping abreast of the latest developments in technologies that support significant parts of the journey. Technical developments move forwards in great leaps and bounds altering the playing field significantly. Drug discovery, with its link to the greater good for humanity, will always be a prime focus for innovative companies. Flow cytometry has been a promising candidate technology for speeding and improving drug discovery processes for some time, and finds use in workflows within the journey. Its ‘traditional’ role in analysing phenotype and cellular assay end points has already seen it used broadly. However, it has been hamstrung from realising its full potential by sheer complexity, necessitating significant training of personnel and leaving reproducibility of results across different operators, instruments, labs and timepoints as something of a lottery.
Developments in the technology over recent years, however, as well as in the antibody technology that plays a critical role in its success – in particular the development of recombinant antibody technology – have overcome these challenges almost entirely. What’s more, clever repurposing of the methodology for novel applications, such as cell sorting, have expanded its utility to further workflows critical to drug development, making it all the more attractive. Now capable of boasting high-throughput, accurate, multiparameter and, above all, highly reproducible results, pharmacological companies are increasing their trust and reliance on this powerful technique to make drug discovery safer and faster. In some case this has shaved months off development schedules – not to mention saving large amounts of capital which would otherwise be sunk in late-stage candidate failures. With an end result of more patients treated and lives saved, it is only a small exaggeration to describe this renaissance as something of a revelation. In drug discovery, it’s time to look again at how flow cytometry can make processes that were previously challenging into humdrum reality.
Volume 22, Issue 1 – Winter 2020/21
About the author
Dr Urmi Roy completed her Ph.D. in Immunology at TU Braunschweig. Here she specialised in mucosal immunology/gut microbiota. She is now Product Manager responsible for the flow cytometry reagent portfolio.
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