Lab Automation Supplement
New technologies aid cell-based assays to come out from behind the shadow of animal testing.
Cell-based assays have become a popular platform for drug discovery. Bill Severson, PhD, a research virologist at Southern Research Institute, Birmingham, Ala., has developed high-throughput, cell-based infectivity assays for pathogenic viruses, including SARS, avian and seasonal influenza, equine encephalitis, and respiratory syncytial virus. These assays help identify potential antiviral drug candidates from large compound libraries.
Severson has thus far secured “several commercial contracts” utilizing the screen. In a typical assay, test cells are cultured for 24 hours into 384-microtiter plates pre-coated with library compounds, followed by a three-day virus infection and end point determination. Cells may be “drugged” after culture as well. Hit molecules are re-tested in serial dilution to obtain dose-response or minimal effective dose.
Severson’s SARS assay, which uses Vero-E6 (monkey kidney) cells, has thus far uncovered several hits from a 100,000-compound library. Calpain-4, a known inhibitor of virus-induced apoptosis, serves as the control. “After that project, we obtained avian influenza, and validated a cell-based screen for it,” Severson says. He has thus far screened a library of one million compounds against bird flu for a “big pharma company” looking for both single-dose compound hits and dose-response toxicity.
The assays go slower than they normally would because of the need to wear protective equipment and operate under biosafety level-3 (BSL-3), which Severson describes as a strength. “Other labs also do ultra-HTS, but they can’t touch virus work.”
Although any number of end points work for infectivity screens, cell death is the usual objective. Numerous marketed assays will provide this information. Severson tests several to see which provide the best signal-to-noise and “Z-value,” a measure of assay reliability. Severson monitors cytotoxicity through several assays, including the CellTiter-Glo luminescent cell viability assay from Promega, Madison, Wis., which monitors cell viability as measured by ATP levels (dead cells don’t have it).
In March, 2007, the National Institutes of Health (NIH) awarded another Southern Research Institute investigator, Gary Piazza, PhD, a grant to develop a high-throughput screen for phosphodiesterase isozyme inhibitors. Piazza is collaborating with Blueshift Biotechnologies, Sunnyvale, Calif., a vendor of instrumentation and reagents for cell-based assays and gene expression studies. Piazza measures levels of cyclic guanosine monophosphate in live cells using Blueshift’s IsoCyte laser scanning fluorimeter. Adapted from semiconductor inspection technology, IsoCyte provides multicolor fluorescence, anisotropy, and scatter images for high content and object-based multiplexed array formats. For the Southern Research Institute collaboration, IsoCyte will measure fluorescence resonant energy transfer (FRET) by anisotropy, which researchers hope will provide more rapid and less noisy readings. FRET experiments are normally done by measuring the ratios of two fluorescent signals as they interact. Since the signals overlap, designing a high-throughput assay is difficult. By relying on anisotropy, the signals are de-convolved more easily and quickly.
As small as proteins
Quantum dots, a recent addition to the toolbox for cell-based assays, are nanometer-sized (about as large as a protein) semiconductor structures that fluoresce when exposed to broad-spectrum light. Quantum dots are made from common semiconductor materials (e.g., cadmium selenide or telluride) coated with a different ceramic (e.g., zinc sulfide) to enhance their optical properties. When illuminated, quantum dots emit fluorescence specific to their physical size and their bulk semiconductor component.
Unlike traditional fluorophores, quantum dots fluoresce through formation of excitons, or electron-hole pairs; fluorescent proteins rely on p- p* electronic transitions, which arise from electrons moving around conjugated double bond systems. In practical terms, this translates into much longer, brighter emission that is suitable for time-gated detection studies. The relationship between a quantum dot’s physical size and its exciton energy, referred to as “tuneability,” makes multicolor assays possible.
Because quantum dots are approximately the size of proteins, cells process them like molecules, not particles. Tanya Vu at the Oregon Health Sciences University, Portland, Ore., conjugates cadmium selenide quantum dots with nerve growth factors. Vu can then observe nerve cells internalizing the quantum dot-protein conjugate, and watch the conjugate travel through the cell to high-growth areas.
Invitrogen, Eugene, Ore., became the leading supplier of research-grade quantum dot products through its acquisition of Quantum Dot Corp. The company’s Qdot nanocrystals have numerous applications in cell biology, including flow cytometry, cell tracing, and in vivo cell imaging, plus standard assays such as immunocytochemistry, immunohistochemistry, Western blotting, fluorescence in situ hybridization, and multispectral imaging. “Qdot bioconjugates are often used as simple replacements for analogous conventional dye conjugates,” says Stephen H. Chamberlain, PhD, senior product manager for particles at Invitrogen. Qdots conjugate easily with protein A, biotin, small molecules, and a range of proteins. The large, ordered Qdot surface area allows simultaneous loading of multiple biomolecules, increasing avidity for targets and general functionality.
Squeezing value from instrumentation
With cost of cell biology rising, maintaining cell imaging and cell-sorting expertise has become too expensive. Numerous user facilities have sprung up, for example, at the University of Utah, Stanford, Vanderbilt, and Yale. Fox Chase Cancer Center in Philadelphia has one of the longest-running NIH grants to operate such a facility. “Our labs tend to be small, so investigators tend not to bring in a lot of postdocs with specialized expertise,” explains Sandra Jablonski, PhD, who manages the Fox Chase Cancer Center’s Cell Imaging Facility.
At the Fox Chase Center, researchers themselves perform the gamut of cell-based studies, including protein staining and location (or co-location), siRNA knockdowns, fluorescence recovery after photobleaching (FRAP), and fluorescence resonance energy transfer (FRET). In FRAP studies, proteins of interest are bleached out, and their recovery is observed in specific areas of the cell.
Naturally, live-cell imaging is a big part of this work. “Everybody wants to do it,” Jablonski says, noting that imaging is increasingly favored over simple plate-reading due to the former’s high information content. “Imaging allows investigators to examine the finer aspects of what’s going on within a cell,” he says. Similarly, researchers are looking for greater value and functionality from cell-viewing and imaging instruments.
Increasingly, cell work demands more than just lighting cells up with a favorite fluorescence-tagging reagent. High-content (the operative buzzword) experimentation requires a high level of automation, data management, experimental versatility, and the ability to resolve events in real-time, according to Ned Jastromb, senior applications product manager at Nikon Instruments, Melville, N.Y.
Nikon is following the modern instrumentation trend in supplying experimentation-enabling devices rather than just instruments. Its recently introduced BioStation CT, a fully-automated cell culture system with built-in microscope and camera, combines image acquisition with automated image analysis of live cells over time. Biostation CT provides protocols for monitoring stem cell differentiation, studying the interaction between cells and substrates, and studying co-cultures in tissue engineering in addition to standard toxicology and pharmacology.
“High-content applications can only be performed accurately in a live cell environment with temporal resolution,” Jastromb says. For example, knowing the precise moment to add a drug or cofactor is critical for assay development. Instead of simply following a time protocol, tools like the Biostation CT can identify the precise moment when cells are ready.
The cancer connection
Much cell-based assay work focuses on cancer, but primary cell culture opens the window into a host of chronic and degenerative diseases, including metabolic and immune system-related disorders. Since cultures of primary cells stop growing after only a few divisions, preserving cells during assays or compound screens is highly desirable, says Mark Roskey, PhD, VP, Reagents and Applied Biology at Caliper Life Sciences, Hopkinton, Mass.
Caliper made its name in the 1990s as a developer of microchanneled analyzers, the “lab on a chip” idea. Today, the company continues in that vein and offers three technologies to support high-throughput, cell-based assays.
The company’s LabChip 3000 drug discovery system shrinks cell-based assays to the dimensions of a microfluidic chip the size of a couple of postage stamps. Cells transferred from microplates travel through and are separated in channels about the width of a human hair and are quantified through laser excitation and a detector. LabChip’s primary advantage over flow cytometry is its versatility and stinginess with valuable sample cells. “It allows you to do cell-based assays on primary cells, which you may not have many of,” says Roskey. LabChip handles almost any type of cell-based assay, including calcium flux and reporter gene assays.
Samples and assays are controlled through the Staccato automation system, consisting of an automatic liquid handler linked to cell culture, plate handlers, plate washers and readers. Almost any cell-based assay can be run.
Caliper’s third enabling technology involves incorporating the gene for luciferase in cells (usually tumor cells), and following the cells and their progeny inside living animals by external imaging. Through the company’s “I-to-I” (in vitro to in vivo) strategy, companies like Pfizer, Sanofi-Aventis, and Merck have developed in vitro screens using Staccato/LabChip, and ported them over to living test animals.
Cell-based assays have been catching on in medical testing as well, with exciting results. Entrepreneurial firms are beginning to commercialize cell-based assays for cancer diagnosis, prognosis, and to predict response to treatment. Eventually, these methods may become routine enablers for tomorrow’s personalized medicine.
But first, they must distinguish between a disease phenotype from what may be numerous genotypes. Of course, gene-based assays are already in use (e.g., the breast cancer drug Herceptin and the her2neu gene), but genes often provide an incomplete picture of response to a drug. “The complexities and redundancies of human biology are beyond the ken of genomics,” according to Robert Nagourney, MD, founder of Rational Therapeutics, Long Branch, Calif.
Rational develops cell-based assays that predict which chemotherapy drugs or combinations will work on a specific patient’s tumor. Its diagnostic product, the EVA programmed cell death (EVA-PCD) assay, uses tissue obtained from biopsy or surgery, and cultured to maintain as faithfully as possible the cancer cell’s native “niche” and physiologic state within the tumor. These cultures are then treated with various chemotherapy agents and combinations to see which drugs work best. Published data from the company’s clinical studies suggest a high degree of correlation between assay results and response to chemotherapy. Similarly, Precision Therapeutics, Pittsburgh, Pa., tests cells from excised tumors against a panel of chemotherapy agents, through the company’s ChemoFx assay and analysis algorithm.
In a study on ovarian cancer conducted with Yale University, patients whose cells were classified through ChemoFx to be “resistant” to chemotherapy averaged nine months before disease progression; those in the “intermediate response” group averaged 14 months, while those found to be “sensitive” to one or more chemotherapy agents remained stable up to publication of the study.
About the Author
Angelo DePalma, Newton, N.J., covers the pharmaceutical, biotechnology, chemical, and healthcare industries.
This article was published in the Lab Automation supplement to Drug Discovery & Development and Bioscience Technology magazines: November, 2007, pp. 26-29.
Filed Under: Drug Discovery
