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Flow cytometry is a tool that can be used to take a heterogeneous cell population and “fractionate” it into several homogenous cell populations. This feature is especially helpful when attempting to identify a drug response in a small subset of cells, even when that subset is out-numbered by cells in a complex biological fluid, such as human whole blood, or in a multi-cellular matrix like human tissue. Flow cytometry was previously considered a major research tool for preclinical studies (albeit a slow one), but its use is expanding into other areas of drug development as the need to analyze complex cellular mixtures, as opposed to cell lines, increases.
“Flow cytometry is a tool that allows drug developers to actually use human blood, not just cell lines, to make drug development decisions,” says Robert S. Balderas, vice president of biological sciences at BD Biosciences, San Jose, Calif. “Cell lines are not going to move out of drug development, but today when you can integrate multicolor applications to look at heterogeneous populations of cells and blood, decisions about drugs could be made based on observations in cells that are closest to human physiology.”
Flow cytometers produced by BD Biosciences have been used in all phases of drug development including clinical trials (e.g. in patient stratification) and other drug registration studies (e.g. in mechanism-of-action studies). For example, one of their flow cytometers, the BD LSRFortessa cell analyzer, can perform high-complexity flow analysis with 15 to 18 different colored fluorescent dyes. But much of their efforts over the last two or three years have been spent developing reagents that measure post-translational events inside cells. Specifically, they have expanded their catalog of phospho-specific antibodies to measure intracellular protein phosphorylation events in primary cells and in whole blood. “The combined use of antibodies against post-translational targets within cells and the use of cell surface marker antibodies provide data on a drug activity in whole blood,”says Balderas. For example, researchers use flow cytometry to determine how a drug that is targeted to a T-cell might affect other blood cells.
Fast flow
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“Flow cytometry systems analyze one sample at a time, which makes the speed of the systems too slow for primary drug screening projects,” says Fred Koller, PhD, chief executive officer, Cyntellect, San Diego, Calif. Celigo, Cyntellect’s new cytometry system has several pharmaceutical applications including validating “hits” from compound screens and detecting drug toxicity in cell-based assays. When used as an analyzer for non-invasive in situ live (adherent and non-adherent) cell imaging, Celigo enables microplate-based brightfield and fluorescent cellular analysis with minimal sample manipulation. “With the Celigo Cytometer, researchers in pharmaceutical and academic cell analysis labs can rapidly and easily obtain high-quality data on any cell type without the need for enzymatic disruption,” says Koller. According to the manufacturer, the instrument’s brightfield and multi-color fluorescence modules and easy-to-use analysis software provide extremely detailed cellular data on each individual cell in the microplate well, and can perform label-free analysis of live cells as well as fluorescence cellular analysis. In brightfield mode, the instrument can perform cell counting and track cellular growth within culture flasks and plates over multiple time points. While in fluorescence mode, it is capable of detecting and analyzing a wide range of fluorescently-stained cells for counting, viability, apoptosis assays, as well as for the detection of high-secreting clones in protein production applications.
Beckman Coulter Inc. (Fullerton, Calif.), has announced a number of innovative products in the flow cytometry arena this year, including the Gallios 10-color, three-laser instrument, which performs highly sensitive, rapid analysis with high resolution at the low end of the scale (for example, easily detecting dimly stained cell populations). “When Gallios is coupled with our proprietary tandem dye technology, it offers significant advantages in the pharmaceutical setting,” said Nigel Llewellyn-Smith, MSc, director of strategic marketing for flow cytometry reagents at Beckman Coulter in Miami, Fla. “For example, the changes in intracellular signaling responses can be very subtle and may be missed by less sensitive flow cytometers.”
Beckman Coulter also offers their CyAn flow cytometry system coupled with the HyperCyt sample plate loader (IntelliCyt Corp., Albuquerque, N.M.). Interfacing these instruments allows drug researchers to use flow cytometry to rapidly perform high-content and high-throughput screening. “Imaging for high-content screening is good, but slow. The CyAn-HyperCyt combination allows researchers to do high-content screening very quickly. A 384-well plate can be analyzed in less than 10 minutes, for example,” says Llewellyn-Smith, who adds that this is a breakthrough for pharma. “We have many large pharma customers looking to us to help them use flow cytometry to rescreen their compound libraries, thus allowing them to rescue compounds that really have potent pharmaceutical effect and which would otherwise be discarded from their development pipelines.”
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Making use of microcapillary flow cell technology, Millipore Corporation (Hayward, Calif.) developed the Guava easyCyte 8HT benchtop flow cytometer. This method reduces the amount of sample and reagent needed, has a low maintenance cost, and takes up very little laboratory space. “In addition to the instrument, Millipore provides more than 40 ready-to-use FlowCellect assay kits to measure chemokine receptor levels, stem-cell phenotypes, cell viability, apoptosis, cell cycle progression, proliferation, and toxicity,” says Rich Pittaro, BSEE, R&D scientist at Millipore. Following sample preparation using Millipore’s FlowCellect assay kits, fluorescently-labeled cells are aspirated into the microcapillary flow cell, then solid-state lasers excite the cells, with each cell emitting a signal that is individually detected by photomultipliers and a photodiode. “Guava flow cytometry instruments and FlowCellect assay kits allow drug developers to obtain multiple end point phenotypic information in a mixed cell population for pathway profiling, target validation, or mechanism of action studies faster and more reliably than with traditional methods such as western blotting, enzyme-linked immunosorbent assays, or immunocytochemistry,” says Pittaro.
It is clear that flow cytometry is increasingly becoming a mainstay in the pharmaceutical world, with applications strategically implemented at points throughout the entire drug life cycle.
About the Author
James Netterwald is president and CEO of BioPharmaComm LLC, a provider of writing, editing, and consulting services to the life science, pharma-biotech, and public relations industries.
This article was published in Drug Discovery & Development magazine: Vol. 12, No. 10, October, 2009, pp. 18-20.
Filed Under: Drug Discovery
