Biochemical drug screens were the norm a decade or so ago, but they didn’t always lead to cures. Targets that looked good in the test tube often failed in animals or people, either because of toxicity or because the drugs were processed differently in the body than they were in a pure chemical interaction. Modern scientists now look to cell-based assays as a drug-development tool that ups the chances of picking a winner.
Although studying medicines in cells is still a far cry from testing in an intact organism, “it’s the first level in biology where you actually have a whole working system,” says R. Terry Dunlay, CEO of IntelliCyt Corporation in Albuquerque, New Mexico. With cell culture, researchers can get as close as possible to a whole organism while maintaining an affordable, high-throughput platform. In fact, Dunlay estimates that more than half of new drug-discovery assays are now cell-based.
But cell-based screens do have their own set of challenges. They are sometimes called “black box” assays, because it’s hard to determine exactly what happened inside the cell between your treatment and the outcome you’re measuring, says Terry Riss, a senior product specialist in cell health at Promega Corporation in Madison, Wisconsin. If all the tumor cells in your assay died, for example, you may have to do further experiments to figure out how the drug actually did them in.
Also, cells are sensitive. Their responses might change depending on how many are sharing a dish, or how much time they’ve had to settle in. Even the best-cared-for cultures are always a heterogeneous mix, so some cells might respond to a drug right away, while others in the same container take time to catch up.
Therefore, it’s important to carefully decide how long to incubate the cells before you make your measurement, Riss advises. “This is one of the most commonly occurring problems that people have,” he says. “They miss the window.” For example, an assay for apoptosis might measure caspase activity. But wait too long, and the cells might already be dead, their caspases degraded.
Here, The Scientist profiles four methods to maximize what cell assays have to offer.
CELL COUNTS, ELECTRICALLY
Method: The xCELLigence plate reader takes advantage of the fact that cells block the flow of electricity. In the reader’s special culture dishes, an array of tiny, circular gold electrodes coats the bottom—to the eye, it looks as if the plates are lined with a thin gold film. “As the cells grow to cover the surface, the electrical impedance goes up. The reading that you get is directly proportional to the surface coverage with those cells,” says user Aykut Üren, a molecular oncologist at Georgetown University in Washington, DC.
Applications: Measuring cell growth and proliferation of adherent cells are typical uses. The method is also convenient for investigating G-protein-coupled receptor signaling, which causes subtle cell shape changes that the xCELLigence can detect. Other xCELLigence users have gotten more creative. Since different cell types create different impedances, Üren screened for molecules that inhibit the ability of tumor cells to invade a layer of endothelial cells (JoVE, 50:e2792, 2011; Oncogene, 31:269-81, 2012). And Michael Smout, a postdoc at James Cook University in Cairns, Australia, adapted the assay to measure motility in parasitic worms, which indicates their general health. As the worms writhe in a well—“like snakes in a bucket”—the impedance signal flickers up and down. Smout calls it the worm-dancing assay and applies it to measure the worms’ susceptibility to different drugs (PLOS Negl Trop Dis, 4:e885, 2010).
ACEA Biosciences, Inc., of San Diego, which markets the xCELLigence, also makes a specialized version for heart cells. The RTCA Cardio (Real Time Cell Analysis) measures how cardiomyocytes beat in culture, providing a readout like an electrocardiogram. Cardiotoxicity is one of the major reasons drugs are dropped or withdrawn from the market, says Matt Peters, a principal scientist at AstraZeneca in Waltham, Massachusetts. He is testing the RTCA Cardio’s potential to catch heart-damaging drugs early in the drug-development process.
Throughput: The xCELLigence can read a 384-well plate in about a minute, says Yama Abassi, vice president for research and development at ACEA.
• No need to label the cells, which might alter their biology, says Reena Halai, a research officer at the University of Queensland in St. Lucia, Australia, who studies G-protein signaling.
• You can follow cells in real time, over days or weeks if desired.
• It’s very sensitive, Halai says.
• No multiplexing.
• With gold electrodes in each nonreusable plate, the assays can get expensive.
• It’s up to you to figure out why impedance changed—whether the cells morphed into a new shape, proliferated, or died, for example.
Tip: “You need to run 101 controls before you start,” Halai says, because the impedance can be influenced by a variety of factors. For example, salt concentration can affect the electric field.
Cost: The xCELLigence reader costs about $200,000, and the plates are approximately $70 each.
GREAT CELL MIGRATIONS
Method: Tumor cells, and several other cell-types, are crawlers. The Oris Pro Cell Migration Assay offers them a distinct space to crawl into. Each well contains a circle of gel, the “detection zone.” When the cells are seeded in the wells, they can fill in the entire surface—except for the space covered by the gel, which blocks their access. But soon after exposure to liquid medium, the gel dissolves, and the cells can migrate into the zone. After giving the cells about a day to crawl in, you can determine how motile they are by quantifying the population inside the detection zone with a plate reader or microscope. For example, researchers might stain the sample with a fluorescent label for actin; the cultures with the most fluorescence inside the circle where the gel had been are the most motile ones.
Applications: The Oris provides data similar to that of the classic scratch assay, in which researchers scrape away some of the cells in a monolayer and watch as surrounding cells refill the area. Scientists can use it to examine drugs’ effects on the cell migration that naturally occurs during development, inflammation, and wound healing. It’s also useful for researchers studying how cancer cells spread; for example, user Shrikanta Chattopadhyay, a physician-scientist at the Broad Institute in Cambridge, Massachusetts, studies how breast cancer cell migration is affected by the presence of other cell types.
Throughput: It takes about 16 minutes to scan a 384-well plate, Chattopadhyay says. He uses the Molecular Devices ImageXpress Micro System automated microscope (See “High on High Content” in this same issue.).
• Chattopadhyay barely has to handle the plates—all he does is add the cells—which minimizes opportunity for error, he says.
• With the circle always the same size and location, the assay is very reproducible.
• Unlike in the conventional scratch assay, the cells are not injured, notes Gopal Krishnan, team leader for cell biology at Platypus Technologies, which makes the plates.
• Because the gel spot starts dissolving as soon as you add the liquid media, you can’t let the cells settle in before starting the assay. Chattopadhyay would prefer to let his cancer cells sit for a day or two in co-culture with cancer stem cells, which influences their motility, before removing the gel.
• Unlike some other motility assays, the Oris cannot measure chemotaxis of cells toward an attractant; it can only measure spontaneous motion.
Tip: Be sure to plate the cells at 90–95 percent confluency, Krishnan says. If you seed too few cells, they’ll have to crawl farther to reach the gel-coated zone. And if you add too many, some may still be suspended when the gel dissolves. In that case, they may land in the detection zone instead of migrating there.
Cost: Plates cost $200–$250 each, depending on how many you purchase.
AN EASIER WAY TO GO WEST
Method: The In-Cell Western assay tells you how much protein you have without all that tedious cell lysis, gel electrophoresis, and sample transfer. Essentially, it’s a whole-well, quantitative version of immunofluorescence microscopy. You fix and permeabilize cells, and stain one or two proteins of choice with primary and secondary antibodies. The secondary is conjugated to a near-infrared tag. Then the LI-COR Odyssey plate reader is used to measure how much infrared fluorescence comes out of each well.
Applications: “The goal of the assay is to quickly and accurately measure the levels of proteins in cultured cells,” says Amy Geschwender, a principal scientist at LI-COR Biosciences in Lincoln, Nebraska. Frequently, researchers use it to measure how much of a protein is phosphorylated. Adherent cells are easiest, but you can use suspended cells by spinning them down onto the plate’s bottom.
Throughput: The Odyssey can read 96- and 384-well plates in about 5 minutes. Because the assay uses large quantities of antibody—which can get expensive—Geschwender recommends it for secondary, rather than initial, drug screens.
• It’s reliable and repeatable compared to classic Westerns, where cell lysis and gel-loading are big sources of error, Geschwender says.
• The assay is sensitive to a large range of protein concentrations, says user Hector Aguilar, an MD/PhD student at the University of Alberta in Edmonton, Canada.
• There is very little background fluorescence in the near-infrared range, so all you see is real signal.
• The Odyssey can read two different near-infrared signals at the same time, from both 700- and 800-nanometer tags. This is useful for directly comparing a phosphorylated protein stained with one antibody to the total amount of unphosphorylated protein labeled with the second antibody.
• You can stop a cellular reaction instantaneously by squirting in fixative. For example, Aguilar was able to analyze the response of muscle cells to drugs after just 20 seconds of treatment (PLOS ONE, 5:e9965, 2010; J Cell Mol Med, 10.1111/j.1582-4934.2012.01625.x).
• Unlike in a gel-separated Western, you don’t see the relative molecular weight of proteins.
• Unless you have an infrared microscope, you won’t be able to localize the signal to a specific cellular location.
• It doesn’t work for frozen samples, because ice crystals may damage the cells.
Tip: The assay requires highly specific antibodies. Antibodies that work well for immunofluorescence microscopy are your best bet for the In-Cell Western, Geschwender says.
Cost: The Odyssey product line costs between $30,000 and $60,000.
FLOW CYTOMETRY AT SPEED
Method: “The mantra is, you never put bubbles in a flow cytometer,” says Bruce Edwards, a professor at the University of New Mexico in Albuquerque. He broke that rule with the HyperCyt system he codeveloped, now sold by IntelliCyt. Normal flow cytometers run one sample, containing cells from a single plate well, through the tubing at a time, followed by a time-consuming wash cycle in between samples. The HyperCyt sucks up samples from hundreds of wells in succession, separating each sample from the next with an air bubble before sending them to the cytometer. The air bubble creates a cell-free gap in the data file, so researchers can read the results from each individual well.
Applications: A HyperCyt system offers the same individual cell outputs as regular flow cytometry: cell counts, cell size, cytoplasmic granularity, and anything you can measure with a fluorescent probe. Checking the binding of new antibodies is a common use, Dunlay says, as are apoptosis assays. It works best with cell cultures grown in suspension.
Throughput: HyperCyt can read 40 samples per minute, from 96- or 384-well plates. (For comparison, typical flow cytometry setups could take more than a minute per sample, says Dunlay.)
• Flow cytometry is a master at multiplexing—doing more than one experiment at the same time. For example, by color-coding five different cell types, then mixing them together, Edwards conducted five screens in one go (ACS Chem Biol, 7:715-22, 2012).
• Instead of filling up the cytometer’s tubing with 100 microliters or more of one sample, the HyperCyt sucks up just a couple of microliters between the bubbles. “It’s a huge money saver,” Edwards says, to cut the volume of high-throughput experiments. Plus, there’s often sample left over to re-run a well or plate if something goes wrong. “It’s saved us many times,” he adds.
• “You can’t beat the sensitivity,” says David Sykes, a hematologist at Massachusetts General Hospital in Boston, who used the University of New Mexico’s HyperCyt services to screen for potential leukemia treatments. “Your range of detection is so great.”
• Cells that clump together will get stuck in Hypercyt’s pump.
• The physical pressure the pump puts on cells can interfere with certain experiments. For example, calcium signals can change in response to the compression, Edwards says.
Tip: Think carefully about the volume of culture—which determines the number of cells—you’ll want to analyze in a high-throughput screen, Sykes says. Even a second more of scan time per well adds up when libraries contain hundreds of thousands of compounds. But if you choose a small volume, and some wells have unexpectedly low cell populations, you may not achieve the cell numbers you want.
Cost: If you already have a flow cytometer, you can add the HyperCyt for $55,000–$65,000. For $125,000–$150,000, it comes with its own cytometer.