Biology for the Information-Age Cell-based Assays

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Biology for the Information-Age Cell-based Assays

Creating High Content From Cells

by Mike May

To study organisms under conditions more closely resembling their natural environment, many researchers are moving from biochemistry-based to cell-based assays. “Cell-based assays provide a highly accurate representation of cellular behavior and offer far more useful information than can be obtained using traditional enzyme or antibody-based assays,” according to Frost & Sullivan’s “Developments in Cell-Based Assays,” which was published in March 2006. The report states: “Since 1990, pharmaceutical and biotechnology companies have been shifting toward the use of cell-based assays over other types of assays, such as biochemical and in vitro assays.” Moreover, cell-based assays provide an opportunity for scientists to study mechanisms in a biological but controllable system.

Transfected cells are automatically detected with IMSTAR’s Pathfinder Nanoscope system.

“The sooner you get in a cell-based assay,” says James O’Connell of ACEA Biosciences (San Diego, CA), “the more likely you are to get biologically relevant information.”

Increasing content

In general, cell-based assays provide researchers with more information. “From a high-content point of view,” says Julie Coughlan of Sigma-Aldrich (St. Louis, MO), “there’s a lot that you can do if you can get inside the cell.” To add to what can be done, Sigma-Aldrich is developing a new assay that can quantify and locate proteins in living cells.

As cell-based assays evolve to deliver higher content, though, more automated processes are required. Javier Farinas of Caliper Life Sciences (Hopkinton, MA) says, “We’re working on a microfluidic approach that saves on reagent usage and pipetting steps.” He adds, “The system will run on most commercial high-content readers.” He expects the resulting products to be available some time this year.

The LabChip microfluidic device from Caliper Life Sciences provides a cell-based GPCR assay. Cells and experimental compounds are mixed inside 50 micron channels that run through the glass portion of the chip. Signals from individual cells are measured through the round detection window (middle right portion of chip) where the microfluidic channels meet.

In moving from microplates to microfluidics, the technology will reduce sample size. “That can be very important for some cell-based assays where the cells are difficult to get,” says Farinas. For example, Caliper worked with Millennium Pharmaceuticals to look at the function of blood platelets in a microfluidic device. “They got answers with hundreds of cells instead of hundreds of thousands of cells,” says Farinas. Moreover, Caliper’s technology will work with most any kind of cell-based assay.

Scientists at IMSTAR (Paris, France) also aim to enhance the content of cell-based assays. IMSTAR’s Pathfinder NanoScope platform captures and analyzes at sub-cellular resolution hundreds of spots, each composed of hundreds of cells, on a slide. “It provides hundreds of micro-experiments on one slide,” says IMSTAR’s Michel Soussaline. He adds, “This system keeps track of quantitative information on every individual cell, enabling the handling of multiple parameters, not just mean values.” Using mean values, Soussaline explains, one can miss differences in cell-phenotypes that appear in just a few percent of the studied cells. The trouble is that the information from a few cells can be crucial in certain kinds of experiment, such as exploring the cytotoxicity of a compound under consideration as a drug.

High-tech tracking

Tracking something often involves attaching a probe to the structure of interest so that it can be seen under visible or fluorescent light. “But hanging fluorescent compounds on molecules changes them,” says O’Connell. “If you can develop an assay without labels, it doesn’t change the binding of the target compound. It works like the natural compound, and you get fewer false positives and negatives.”

An example of such technology, the ACEA RT-CES microplate system, requires no labels. In addition, this system for cell-based assays provides feedback in real time. “From the moment you first pipette cells into the wells, you get a reading of what’s going on with them,” says O’Connell. If a gene-expression assay produces a spike, the system can show when the spike took place.

This system can be applied to many situations. O’Connell notes that some scientists use this as a quality-control step with cultured cells. “If you start seeing wild variations,” he says, “then maybe it’s time to go back to the freezer and get new cells.” In general, though, O’Connell says this technology can be applied to most any cell-based assay.

Figure 2. IMSTAR’s technology can keep track cells that get transfected with GFP in groups of cells in spots on a microarray (a). A magnified view (b) shows transfected (green) and non-transfected (blue or red) cells, and the transfected cells can be automatically detected (c). Click to enlarge.

ACEA is also working on a prototype system that will track the invasion and migration of cells. “Invasion is important for cancer cells, “says O’Connell. “You will be able to use this cell-based technology to look for compounds that stop cancerous cells from invading other cells and tissues.”

In some situations, just tracking a compound might not be enough. Instead, scientists might want to know if two biomolecules interact, which can be monitored with fluorescence resonance energy transfer, or FRET. In very simple terms, FRET relies on donor and acceptor fluorophores that are attached to the two items of interest, and if they get close enough, a stimulus excites the donor fluorophore, which passes energy to the acceptor, which releases light that verifies the interaction. Scientists at Cisbio (Bedford, MA) use a type of FRET in homogeneous time-resolved fluorescence, or HTRF. Although this approach is not new, its application in cell-based assays is growing.

Conventional FRET often generates lots of background fluorescence in cell-based assays. “HTRF takes advantage of a ratiometric measurement,” says Chris Harbert of Cisbio. This corrects for background issues. Krista Steger of Cisbio says, “We started with the premier cell-based assay — cAMP — and now we’re going on to other assays.”

Digging out better drugs

“A few years ago,” says Berta Strulovici of Merck (North Wales, PA), “no one thought cell-based assays would have the value that they do in drug discovery.” The benefits of cell-based assays, such as telling if a compound penetrates cells and hits the right target in a physiological environment, cannot be denied. According to Strulovici, more than half of the assays being developed at Merck now employ intact cells. “This covers all sorts of therapeutic targets, G-coupled protein receptors, enzymes, protein-protein interactions, and so on,” says Strulovici.

In some cases, Merck collaborates with academic researchers to develop new assays. Gideon Dreyfuss of the University of Pennsylvania identified a protein survival motor neuron (SMN) related to spinal muscular atrophy. Now, Dreyfus and Merck are collaborating. Dreyfus provides his SMN assay, and Merck provides the technology to screen millions of compounds against this target. “Spinal muscular atrophy is a devastating disease without a known cure,” says Strulovici. “The underlying biology is complex and not easily amenable to drug discovery. Combining innovation from the Dreyfuss lab with Merck’s approaches to early lead identification may provide the ingredients needed to develop a breakthrough.”

Figure 3. Invitrogen’s GFP fusion protein TR-FRET kinase assay can reveal phosphorylations of proteins by using an antibody labeled with terbium. Click to enlarge.

Other companies also aim cell-based assays at drug discovery and development projects. For example, Liz Gardiner of Kalypsys (San Diego, CA) says, “We focus on assays that can be used in primary and secondary screening for metabolic diseases, pain, inflammation, and cancer.” To help with cell-based screening, Kalypsys designs assays in 1,536-well formats. “That makes it possible to run a million compounds in a day or two,” says Gardiner. Kalypsys scientists also employ cell-based assays to find biomarkers to use as indicators of efficacy and safety in clinical trials.

Scientists at Invitrogen also use cells as indicators early in drug discovery. “In cell-based assays,” says Invitrogen’s Brian Pollok, “we focus primarily on druggable targets, like G-protein coupled receptors (GPCRs), protein kinases, and nuclear receptors.” Invitrogen currently offers 46 off-the-shelf pharmacologically validated assays for GPCRs.

Invitrogen developed cell-based assays that reveal specific post-translational modifications. When a protein labeled with GFP gets phosphorylated, it binds to a phosphorylation-specific antibody labeled with terbium. The terbium serves as a donor in time-resolved FRET, which lights up the GFP. “This method allows one to quantify how compounds affect protein phosphorylation or ubiquitination of physiological substrates in situ, in a manner that is compatible with high-throughput screening,” says Pollok.

Taking on tissues

Cell-based assays can also reveal the right drug for a particular patient. For example, a biopsy from a patient tumor could be grown and then tested against a panel of chemotherapeutics. To make this work, though, the assay must quickly test many compounds. Jay Leng of Cell Biolabs (San Diego, CA) says, “We developed a 96-well, in vitro, tumor-sensitivity assay. A doctor can do a biopsy and then send it to a lab where it can be grown and mixed with chemotherapy drugs.” The assay’s results — available in about a week — show which drug most effectively attacked the patient’s tumor.

In addition to assays that personalize treatments, cell-based assays will move increasingly into imaging. Coughlan of Sigma-Aldrich says, “It will soon be routine for people to do single-cell imaging. Also, whole-animal imaging will become much more prevalent. That will be the next wave.” She adds, though, that these assays will require new tools, such as different imaging agents and dyes.

Testing compounds on individual patients, imaging, and other high-content approaches to cell-based assays will also increase the amount of data that needs to be analyzed. A high-content image can easily be a few megabytes in size. Take a few images for each well in a microplate, and a scientist’s computer will soon get swamped. So as cell-based assays improve, the bioinformatics must improve as well. Otherwise, scientists will reap mountains of information with no way to comprehend it.