Editorial Article
Monday February 16, 2009
by Caitlin Smith
Measuring cell proliferation
—or the related cell viability—would seem to be simple at first glance: how many cells are dead or alive, or in the process of dying? A second look tells you that such measurements aren’t so simple. For example, most (but not all) proliferation assays use labels that become incorporated into the cell, which may be physiologically disruptive. “These labels can interfere with the normal physiological behavior of the cell,” says Manfred Watzele, R&D director at Roche Applied Science. “Since these assays are often destructive to the cells, they are usually endpoint assays.”
Other factors may affect the quality of your assay as well. “The biggest challenge is choosing the appropriate biomarker of viability and the assay chemistry to match the conditions of the experiment,” says Pam Guthmiller, strategic marketing manager for Promega. “[For example,] when working with precious cells such as primary or stem cells, researchers are limited by the number of cells used per well. Colorimetric assays are not sufficiently sensitive for limited cell numbers without compromising data quality. Compound libraries often have certain classes of compounds that have autofluorescence, which can lead to high background in a fluorescent-based assay. To account for these issues, one must carefully consider assay chemistries that allow for confirmatory assays to be performed in the same well.” Recent releases in cell proliferation assays show how they offer different strengths for different experiments.
Non-toxic timeline assays
Though a lot of cell proliferation or viability assays are meant to take a snapshot of living cells in culture, for example, others give you the ability to evaluate cell proliferation dynamically or even continuously. BioVision’s new VisionBlue™ Quick Cell Viability Assay uses the redox dye Resazurin, which fluoresces brightly upon reduction by cells that are metabolically active. BioVision claims the assay is more sensitive than another popular dye, Alamar Blue, and “needs only as few as 100 cells,” says Gloria Zhang, product manager at BioVision. And “since the reagents are non-toxic, researchers can read plates at various times to follow cell proliferation dynamically.”
If labels are causing problems in your experiments, consider Roche Applied Science’s xCELLigence System, which lets you monitor adherent cell lines in real time using no label. The system allows noninvasive, continuous monitoring of impedance. “Unlike in current colorimetric or fluorescent assays, the cells can be used for further analysis after monitoring the proliferation,” says Watzele. “Unlike [methods that] produce endpoint measurements only, the xCELLigence system also allows a continuous measurement of the cells throughout the whole growth phase or during assays involving compound treatments.” The result is information about the time course of effects rather than simply an endpoint.
Fast pathways to large amounts of information
The advent of high-throughput cell assays is making its way to the area of cell proliferation and viability. Multiplexing, or using different fluorophore tags to perform several experiments simultaneously, is another way to generate a lot of data. Promega’s new CellTiter-Fluor™ Cell Viability Assay reveals live cells using a labeled peptide substrate. An intracellular protease cleaves the label and activates the signal. “Signal intensity is directly proportional to protease activity. The amount of fluorescence produced correlates with the number of viable cells,” explains Guthmiller. “The reagent is not lytic so additional assays can be followed in the same well of cells.” She says that “microplate-based cell viability assays are the most common methods for measuring proliferation, especially for higher-throughput applications, when many plates and potentially thousands of wells are used in the experiment.” Promega’s new assay is sensitive enough to detect about 40 cells per well, and can be miniaturized to 1536-well format. “In addition, one can easily multiplex with other measurements, such as reporter gene assays, caspase assays, or cytotoxicity assays, to obtain more biologically relevant information from each well of cells,” says Guthmiller.
Another way to acquire a large amount of information quickly is with Millipore’s cell proliferation applications for high content analysis (HCA) and flow cytometry. “Our HCA assays enable multi-parameter single cell analysis by staining cells with multiple, target-specific antibodies,” says Matt Slater, product manager for protein research at Millipore. “These assays allow scientists to determine the amount of cell proliferation, as well as the mechanisms that are affecting it. In addition, scientists can screen for the toxic effects of various compounds and determine how these effects influence cell proliferation.” Millipore offers new cell proliferation kits designed for use with their Guava bench-top flow cytometers. For example, their ViaCount Assay distinguishes viable, dead, and apoptotic cells using two DNA-binding dyes: a nuclear dye stains nucleated cells, and the viability dye stains dying cells. The HCS210 assay includes detection for Ki-67, a cellular proliferation marker. Slater adds that HCA and assays using benchtop flow cytometry “allow for a faster, easier, and more accurate measurement of cell proliferation. As the science continues to evolve towards a systems approach, researchers will want to know both whether cells are proliferating, and the mechanisms causing them to proliferate, or inhibiting them from proliferating.”
Toward therapies with stem and immune cells
Both stem and immune cells are special systems for which proliferation is of particular importance. Optimizing proliferation assays for these cell types could have advantages for research and clinical applications in the future.
Sigma-Aldrich
offers a newly expanded line of membrane-intercalating fluorescent tracking dyes for tracking cell proliferation based on the decrease of fluorescence that occurs with 8-10 cell divisions after labeling. Their newest far-red dye is CellVue® Claret. “The extent of proliferation can be assessed based on the principle that each round of division gives rise to daughter cells with half the fluorescence intensity of the preceding generation,” says Leigh Gaskill, market segment manager for antibodies at Sigma-Aldrich. “Proliferation monitoring can be initiated immediately post-staining and gives results comparable to those obtained with visible-emitting proliferation dyes. Proliferation analysis with CellVue® Claret also offers ... the potential for simultaneous proliferation monitoring of multiple cell types (stimulator vs. responder cells; regulatory vs. effector T cells).”
Gaskill says that the method of using tracking dyes “can be combined with immunophenotyping to follow the evolution of complex immune responses at the single cell level, providing critical details impossible to obtain with bulk assays such as tritiated thymidine or MTT.” These details may speed the development of stem and immune cell therapies, which Gaskill sees centered on several key questions. For example, “how does failure of precursor cells to properly balance proliferation vs. differentiation contribute to immunodeficiencies, autoimmune diseases, hematopoietic cancers, etc?” asks Gaskill. “How do ex vivo transduction and/or expansion conditions affect the proliferation (and differentiation) of input cells and therefore the final composition and function of the cell product to be reinfused? Do reinfused cells home to, engraft at, and/or proliferate at target sites within the body? What happens at non-target sites?”
Today’s tools are beginning to answer such questions. For example, multiplexing using multiple colors and probes per cell with flow and image cytometry is yielding more information per experiment than ever. In addition, Gaskill notes the valuable “synergies obtained when proliferation tracking dyes are combined with phenotypic and genotypic probes to determine the fate of a particular cell or cell type within a complex biological environment.” For example, says Gaskill, “in vitro studies that combine proliferation tracking dyes with a conditionally expressed genetic tag, functional knockdown with siRNA, or similar tools offer a powerful approach for studying the details of cellular control systems and identifying new therapeutic agents that target them.” The newer technologies for cell proliferation assays will surely be eliciting more intriguing information than ever before.