Cell-based assays, important systems in their own right for testing physiological processes, have become a powerful means to predict the outcome of in vivo assays, as well. This is especially vital for the preclinical development of drug candidates. “Rapid advancements in cell-based assays have led … an increasing number of pharmaceutical drug researchers to employ cell-based assays in large screening programs,” says Gopal Krishnan, manager and team leader in cell biology at Platypus Technologies. “New cell-based assay technologies have the potential to lead to efficient and reliable drug screening processes.”
But with great power comes great responsibility. The forethought invested in establishing an appropriate cell-based assay with ideal conditions for your system will pay greater dividends in the future. “When setting up a cell-based assay, it is critical to understand the biology of the model system being studied,” says Kevin Kopish, strategic marketing manager at Promega. “What cell type is relevant to my study? How do these cells grow or behave? Do they express the necessary proteins or have intact cell signaling pathways?” Perhaps more important is a complete awareness of the effects that your experimental manipulations have on the system. “Transfection of a gene of interest into a cell line can have a variety of effects, from toxic effects from the reagents to changes in the transcription and translation of genes at typical rates for the cells being studied,” says Kopish. “It is important to use tools to study biology that have as little impact on that biology as possible.”
Cells on the move
Cell-based assays are particularly well-suited for studying how cells grow and move in certain environments—manipulations that are difficult to study in vivo. Cell Biolabs offers a unique technology to study anchorage-independent cell growth, which is usually assayed by growing cells in a 3-D soft agar medium. “Neoplastic cells in this environment can form colonies that can be visualized under the microscope after about three weeks,” says Ken Rosser, director of marketing and sales at Cell Biolabs. However, besides the three-week delay, the assay also involves manual counting of cell colonies. The Cell Biolabs Cell Transformation Assays improve upon the traditional soft agar assay. “No tedious manual counting of colonies is required,” says Rosser. “Colonies are quantified with a fluorescent dye that is much more sensitive than the naked eye. This allows the total assay time to be decreased from three weeks to just seven days. In addition, we developed a modified soft agar medium that allows viable, intact cells to be isolated following the assay to facilitate further downstream analysis. Viable cells cannot be isolated from standard soft agar assays.”
Cell migration assays represent a sizable proportion of cell-based assays, with the scratch assay being a model for cell migration during wound healing. The scratch assay is popular partly because of its relative simplicity, but it has limitations when looking at the biochemical processes during wound repair. Commonly, a pipette tip is used to make a scratch in a monolayer of cells—attractively simple, but lacking in reproducibility. EMD Millipore’s Cell Comb™ Scratch Assay is designed to remedy this. “The Cell Comb™, a tool for creating multiple scratch wounds, has been optimized to apply a high-density field of scratches to maximize the area of wound edges while leaving sufficient numbers of undamaged cells to migrate into the gap,” says Jun Ma, product manager for cell-based assays in EMD Millipore's Bioscience division. “This form of high-density wounding creates a high proportion of migrating cells to quiescent monolayer cells, which permits sensitive detection of the biochemical events occurring, specifically in the migrating cell population.”
An alternative form of the scratch assay is Platypus Technologies’ Oris™ Pro migration assay, which uses nontoxic biocompatible gel to form a cell-free zone. “The gel dissolves to reveal the detection zone and avoids the use of wash steps or additional reagents,” says Krishnan. “This technology enables compatibility with automated liquid handling equipment for cell seeding and allows unrestricted access to cells throughout the experiment.”
Another gel-type cell assay is offered by EMD Millipore. Its QCM™ Gelatin Invadopodia Assay kits facilitate studying invadopodia—protrusions of localized protease activity—in cancerous cell types, and podosomes in nonmalignant cells. Traditionally, invadopodia and podosome formation are visualized by plating cells onto a fluorescently labeled matrix and then looking for loss of fluorescence, which indicates localized protease activity and matrix degradation. “Our new QCM™ Gelatin Invadopodia Assays provide a simplified and standardized protocol for affixing a thin layer of prelabeled fluorescein (green) or Cy3 (red) gelatin to a glass substrate as well as reagents for co-localizing the actin cytoskeleton and nuclei with degradation sites,” says Ma. “The kits enable visualization of degradation produced by normal and malignant cell types. This degradation can be quantified by image analysis methods and used to track proteolytic time course studies as well as modulator effects on invadopodia formation and ECM [extracellular matrix] degradation.”
Glowing reporters and live cells
Many cell-based assays rely on fluorescent or luminescent reporter gene assays as reliable readouts. For example, Promega’s new NanoLuc™ Luciferase is a reporter enzyme engineered to be much brighter than other forms of luciferase. In addition, it uses a novel substrate, furimazine. “The high-intensity luminescence of the NanoLuc enzyme combined with low autoluminescence of the furimazine substrate allows the sensitive detection of low levels of luciferase expression in experimental systems where other reporters can fail,” says Kopish. “The Nano-Glo™ family of assay reagents support[s] both lytic and live cell applications.” Promega also offers the new luminescent-based ADCC Reporter Bioassay for studying antibody-dependent, cell-mediated cytotoxicity (ADCC).
For visualizing proteins in real time using live cells, EMD Millipore’s LentiBrite™ Lentiviral Biosensors rely on the transduction of cells with fluorescent GFP (green fluorescent protein)- or RFP (red fluorescent protein)-tagged proteins. “Compared to other nonviral transfection methods, lentiviral transduction offers higher transfection efficiency and more homogeneous protein expression, even for traditionally hard-to-transfect primary cell types, such as neurons and stem cells,” says Ma. “These biosensors have been validated for use with fixed and live cell fluorescent microscopy.”
Reporter assays are also instrumental in giving readouts of intracellular signaling pathways. Qiagen’s Cignal Reporter Assays, offered as dual-luciferase (firefly luciferase and Renilla luciferase) or GFP reporter genes, come in a DNA construct format or a lentiviral format. The company’s new Cignal 45-Pathway Reporter Array assays 45 signaling pathways. “The Cignal 45-Pathway Reporter Array enables researchers to simultaneously quantify activity of 45 different cell signaling pathways in a single experiment,” says Vikram Devgan, director of R&D at Qiagen. “The array has predispensed dual-luciferase reporter DNA constructs for 45 specific pathways. Researchers can use their choice of cell line and transfection reagent to easily pinpoint the pathways that are perturbed by a specific gene or drug.”
Quantification of fluorescent or luminescent reporter assays requires a means to measure the signal output. BMG Labtech’s PHERAstar FS is a microplate reader that is unique in its method of reading signals. “The PHERAstar FS can perform cell-based microplate assays that were not possible before,” says EJ Dell, international marketing director at BMG LABTECH. “When measuring live cell-based assays on a microplate reader, it is necessary to measure from the plate bottom so that a microplate lid can be used to prevent cell-contamination and to limit evaporation. Flexible fiber optics are usually used to measure from the microplate bottom. The PHERAstar FS, however, was designed with a Direct Optic Bottom Reading approach that is analogous to a microscope. The result is a higher overall signal in live cell-based assays compared to instruments that use fiber optics.” Such a system is not as sensitive as confocal microscopy, but it takes less time, and it allows higher throughput, while being gentler on the health of the cells.
Cells as reagents
Kopish notes a growing interest in complete turnkey cell system assays. “This means assays that include cells or cell lines not only as critical reagents but as ‘cells as reagents,’ wherein the cells are provided in a frozen, ready-to-use state, which does not require propagation or banking,” he says. “We are developing multiple such cell systems that include ‘cells as reagents’ and are working to provide cells in frozen thaw-and-use formats, which perform better than standard cell cultures.” For example, traditional ADCC assays can be highly variable because the effector cells, peripheral blood mononuclear cells (PBMCs), must be purified from blood. Promega’s genetically engineered effector cells save time and—more importantly—reduce the variability contributed by purified PBMCs. “The effector cells in the frozen thaw-and-use format provide a much simpler model system with greatly reduced variability,” says Kopish. The reduced variability of complete turnkey cell assay systems, including cells as reagents, would be advantageous in screening applications—both in pharma and in the everyday lab.
The image at the top of the page is from EMD Millipore's QCM™ Gelatin Invadopodia Assay (Green).