By Alan Dove, PhD  An artist’s rendering shows the optical refraction of light through an adherent cell, the basis of Corning’s Epic label-free assay system. (Source: Corning) |
When high-throughput screening came into vogue in the 1990s, it divided biology into distinct tiers. Primary screens, which involve thousands or millions of tests per day, required biochemical assays that were homogeneous, fast, and easy to quantify. Cell-based assays, with their finicky environmental requirements, slow incubations, and well-to-well variability, were relegated to lower throughput secondary screens.
Not anymore. "The cell-based assays have had sort of a resurgence over the last five-plus years, and have really been growing in frequency in some of the laboratories," says Ron Verkleeren, business director for the Epic screening system at Corning in Lowell, Mass. Verkleeren adds that boosting the throughput of cell-based assays, and even using them as primary screening tools, "is a very hot topic in both the industry and academia."
Several scientific breakthroughs have driven the new trend. After discovering that many critical signaling pathways converge on a few types of molecules, many drug developers narrowed their efforts to a handful of cellular targets. Meanwhile, researchers developed new quantitative assays that could be used in live cells, making automation easier and yielding much faster results. "The more people have confidence in these assays and the easier they are to use, they then skip the step of the biochemical assay," says Martina Bielefeld-Sevigny, PhD, vice president for reagents and solutions at PerkinElmer in Waltham, Mass.
Off the shelf
 A technician operates a Corning Epic label-free high throughput screening system, which can be used for either biochemical or cell-based signaling assays. (Source:Corning) |
Equipment makers are feeding the trend toward higher-throughput cellular assays with a growing number of kits, reagents, and screening systems. Nearly every major laboratory supplier has at least a few products for automated cell signaling studies, and the biggest vendors have jumped into the field with both feet.
At the American Society for Cell Biology meeting in December, Thermo Fisher, also of Waltham, Mass., presented a series of workshops and product announcements related to cell-based assays. The lab supply giant now offers thermoresponsive surfaces for culturing cells, and a line of media, sera, and supplements for growing somatic stem cells.
Not to be outdone, Perkin-Elmer had some announcements of its own. "At the ASCB meeting we [announced] a range of new AlphaScreen SureFire [cell signaling] kits. We are launching 11, and that brings us up to ... 36 kits now in this area," says Bielefeld-Sevigny.
The AlphaScreen kits build on PerkinElmer's popular AlphaLISA technology, which allows researchers to perform ELISA-style antibody sandwich assays directly in cell culture wells. The no-wash protocol is easy to automate, and the new kits are designed to measure the activities of numerous kinase pathways quantitatively. The company recommends reading the results with their own luminescence reader, but Bielefeld-Sevigny says other scanners will also work.
Smaller suppliers are also in the game, often selling assays that offer unique advantages. Li-Cor, in Lincoln, Neb., for example, has established a healthy niche for its proprietary In Cell Western technology. "The technique is actually quite simple, it's immunohistochemistry, and it's just using two-color fluorescence to do the analysis," says Mike Olive, PhD, the company's vice president of science and technology for biotechnology.
In a typical experiment, researchers use one antibody to measure a particular signal, such as phosphorylation of a receptor, and another antibody with broader activity to determine the number of cells in each well. The antibodies carry labels that fluoresce in the infrared region, minimizing the visible-range autofluorescence that can confound other assays.
 The Roche xCelligence cell-based screening system measures electrical resistance to detect changes in cell shape. (Source: Roche Disgnostics) |
"In the end, we look at the ratio of the two channels, or the two signals," says Olive, adding that "it gives you a quantitative number for what's actually happened, because while the number of cells may vary somewhat from well to well, the ratio always stays the same in a particular sample." The technique requires Li-Cor's proprietary infrared plate reader, but that does not seem to have dampened interest in it; Olive says a recent webinar on the system drew more than 600 attendees.
Other companies are focusing on assays that don't require antibodies, such as the Cignal Reporter system from SABiosciences in Frederick, Md. Using DNA constructs with transcription factor response elements upstream of either GFP or luciferase reporters, the assay can measure a wide range of intracellular signals. The GFP constructs are designed for single-cell assays, while the luciferase constructs are optimized for high sensitivity.
The company has also developed a prepackaged version of the assay on 96-well plates, with the DNA constructs already dried onto the plate surface. "In a single experiment [one] can try out different conditions and get the biological response of any particular stimulus ... and their effect on 10 different signaling pathways," says Vikram Devgan, PhD, R&D lead for cell-based assays at SABiosciences.
Big boxes, little sensors
Besides novel assays, equipment developers have been working on new types of gear for cell-based analysis. Many of these tools come from dedicated efforts to automate cell biology, but some of the new devices were originally intended for biochemists.
 Calcium response kinetics of a GPCR agonist in a multiplex GPCR calcium assay. Each trace in the figure represents the calcium response of a single concentration of the agonist in the assay. (Source: Ke Liu, NIH) |
Corning's Epic system falls into the latter category. The centerpiece of the machine is a biosensor that uses light refraction to measure very small changes in a sample solution. "The concept that we set out to develop with this biosensor was just that ... we could measure whether or not binding occurred in a biochemical assay," says Corning's Verkleeren. But he adds that "in the process we made this discovery that really wasn't obvious ... to get this kind of detailed [cellular] information just by measuring this aggregate movement of mass, mainly proteins, that takes place within a cell."
With the new system, researchers can use changes in cells' optical refraction to track changes in their intracellular structures without labeling. Properly designed controls can indicate which signaling pathways are causing the perturbations, with surprising precision. "It is very, very specific, and really the magic here is how tightly regulated these cells are, they're just not random amoebas, the biochemical reactions that take place within a cell are actually very tightly regulated," says Verkleeren.
Other label-free screening platforms also capitalize on cells' minute structural rearrangements. In the recently introduced xCelligence system from Roche Diagnostics in Penzberg, Germany, a plate of minuscule electrical sensors detects changes in cell shape. "The device is an instrument measuring the impedance, so as a cell is growing, the resistance will grow. If a cell is receiving physiological changes, the resistance will be different from the state before. All this .. can be measured and interpreted," says company spokesman Burkhard Ziebolz.
The first xCelligence system used a single sensor, or e-plate, but another model now uses a six-well array of the electrical culture surfaces, "so one can expect a sixfold [increase in] throughput and of course we are thinking of increasing the capacity further," says Ziebolz. The higher-throughput systems should be available later this year.
While the Corning and Roche machines are general-purpose screening tools, other systems are much more specialized. Many pharmaceutical companies have focused laser-like attention on a single class of signaling molecules, the G-protein coupled receptors (GPCRs), and measuring the activity of GPCRs has now become its own niche within cell-based assays. Indeed, at least two companies now offer dedicated high-throughput screening systems for exactly this assay.
In the U.S., the fluorometric imaging plate reader (FLIPR) systems from Molecular Diagnostics have become the standard for GPCR assays. Hamamatsu, in Hamamatsu City, Japan, offers a competing product line, the functional drug screening system (FDSS), but the company has had a hard time penetrating the U.S. market. "Hamamatsu is a traditional engineering company, very engineering focused, so we're not very good at marketing," says Shouming Du, Hamamatsu's product manager for FDSS in the U.S. Nonetheless, he urges researchers to compare the two systems closely before choosing one, as their designs differ considerably.
Regardless of the system investigators use, they need to remember that cellular assays still differ from biochemical ones. "For biochemical assays a lot of people are used to homogeneous assays where they just take a reading on the whole well, whereas for cell-based assays ... it's pretty important to make sure there's not a whole lot of heterogeneity inside of the well, so that you can count on a single cell reading to reflect the entire population," says Jeffrey Hung, director of marketing for SABiosciences.
The other major challenge, of course, is maintaining the cells themselves. With more sensitive tools, researchers are discovering just how hard it can be to get reproducible results, especially when cell lines have grown through numerous passages. "Where things can go wrong in these assays is rarely ... at the detection level, it's really in the cell culture," says PerkinElmer's Bielefeld-Sevigny.
As cell signaling assays become more popular for primary high-throughput screens, some experts also see them finding a place higher up in the applied science pipeline. "It would be good if we really could come to a process for the pharma industry and for the cosmetics industry and could replace a big part of the animal experiments, that is a big target that we have," says Ziebolz.