Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
For many cell biologists, using tags and labels to dissect and characterize cellular components and targets offers important advantages. Cellular tags and labels have helped scientist to better understand many key events, but a question remains as to whether these reporters influence native cellular states and measured results.
Label-free technologies are an alternative approach used by cell biologists and pharmaceutical companies alike. With this approach, researchers can study live cells without worrying that a fluorescent tag is causing secondary effects, for example. Likewise, pharmaceutical researchers are always seeking ways to screen drug candidates in cells that are as close to physiological conditions as possible. Here are some currently available label-free technologies that researchers can use in live-cell assays.
Measurements using electrical impedance
ACEA Biosciences offers label-free assays with xCELLigence Real Time Cell Analysis (RTCA) technology, which uses measurements of electrical impedance to assess the extent of cell attachment to the bottom of a customized culture plate. Electrodes incorporated into the bottom surfaces of the company’s single-use, disposable, microwell E-plates measure electrical impedance in real time. As a result, changes in cell number, attachment and morphology can be measured. The xCELLigence RTCA Cardio system uses impedance to monitor the spontaneous beating of cardiomyocytes—before and during treatment with cardiac labiledrugs, for example. The company’s second-generation CardioECR system also includes field-potential electrodes to monitor electrical activity and provides a stimulatory pacing function. Researchers are also studying cells “from patients with heart disease, to look at the morphology, beating activity, and electrical activity of cardiomyocytes,” says Leyna Zhao, global marketing manager at ACEA Biosciences. Such assays are valuable to pharma for their predictive power, “screening for compounds with adverse effects on human cardiomyocytes, so that they won’t end up in a clinical trial or on the market,” she says.
With a growing number of cancer drugs in the form of antibodies or immune-cell therapies, researchers are looking for assays that can test the efficacy of antibodies or immune cells for specific cancer-cell types. ACEA Biosciences’ technology also provides an automated homogeneous screening platform that captures the kinetics of immune-cell killings of the target cancer cells. “Researchers want an assay to predict whether or not their engineered T cells, such as CAR-T cells, with specific antigen receptors to recognize tumors, have good efficacy toward cancer cells,” says Zhao. The assay uses electrical impedance to measure cell death when immune cells, such as T cells or NK cells, are added to tumor cells attached to the electrode plates. Because the immune cells don’t attach, they have no effect on the electrical impedance, which is used to measure the extent of tumor-cell death.
Ptychography measurements
Ptychography is a method that uses phase-shift microcopy data to generate high-fidelity, high-contrast, quantitative images. The UK-based company Phase Focus uses ptychography in the Phasefocus VL21, a label-free live-cell imaging and assay system. Tracey Zimmerman, vice president of global sales, says that unlike traditional phase-contrast microscopy, ptychography is gentler on the cells. “Effects from illumination are negligible due to the very low power requirements of the 635-nm laser diode, which is many orders of magnitude lower than for a confocal microscope, for example,” she says. Another way that the company’s method differs from conventional phase-contrast microscopy is that it can gather data on cell thickness and cell volume. Such data are used in smooth-muscle contraction assays in the development of drugs for asthma and Chronic Obstructive Pulmonary Disease (COPD).
Pixel measurements
Digilab offers another microscopy-based system. The MIAS-2™ multimode microscope, along with the eaZYX™ Image Analyzer imaging software, is a digital imaging system that uses a high-resolution CCD camera to create live-cell images without using labels. “It does this by counting and recording pixels,” says Digilab CEO Sid Braginsky, “From that, it can image things such as cell movements, changes in morphology, cell communication or regenerative tissue engineering.”
The system is based on the ability to pixelate images taken at high resolution and high magnification. To create images, the system uses the concept of “scale space,” which identifies objects by their spatial, spectral and temporal characteristics. “As long as the image is picked up as a pixelated video image,” says Braginsky, “the MIAS-2 can tell you the size and volume of a cell, what is happening within cells and how cells communicate.” The MIAS-2 is also both high-content and high-throughput—it can measure plates in up to 1,536-well format, and it integrates with robotics systems for automated cell-based assays.
Labeling metabolic products
Instead of labeling cells, Seahorse Bioscience (recently acquired by Agilent Technologies) detects metabolic products by using fluorescent-based, solid-state sensors to detect real-time changes in concentrations of media analytes in its cell-based assays. The methodology of the company’s systems is based on two measurements: the cells’ rate of oxygen consumption and the cells’ rate of proton production. As cells use energy, they use up oxygen in the media, which the instrument detects by monitoring the signal of an oxygen-specific fluorophore. Likewise, as cells undergo glycolysis, they release protons into the cytoplasm, which the technology measures with a proton-specific fluorophore.
These two measurements are used in Seahorse’s XFp Cell Energy Phenotype Test, which can identify the metabolic status of cells or can simply give information about cell health. A 2D plot of the two measurements yields four quadrants, which represent four different types of cellular metabolism: aerobic (mainly post-mitotic cells, such as neurons), energetic (such as proliferative cells or activated macrophages), glycolytic (mostly unique to cancer cells) and quiescent (stable cells that are not highly active, such as memory cells). The fact that different cell types have distinct energy phenotypes can be very useful. “This assay lets you phenotype cells metabolically, literally in a few minutes,” says Seahorse chief scientific officer David Ferrick. “From there, you can look deeper into pathways or test whether a drug pushes the cells more toward another phenotype.” Seahorse recently released its XFp instrument, which is designed for researchers who want to perform assays like these quickly and easily. (Metabolic experts will be interested in the company’s flagship instrument, the XFe.)
Immunotherapy for cancer also is taking advantage of energy phenotyping. “We believe that different metabolic programs push cells into different lineages,” says Ferrick. “It may be a metabolic program that determines how effectively T cells can kill tumor cells.” In a collaboration with Mike Milone at the University of Pennsylvania, Seahorse is trying to engineer patients’ T cells to make them more targeted, with higher killing activity, for cancer cells. The partners also believe that metabolic programs affect how well immunotherapy works for different cancer patients—some patients have a lasting protective response, while others experience a relapse. “Memory cells in the quiescent quadrant of the energy-phenotyping test may play a role in these lasting responses,” says Ferrick.
Today’s cell-based assays can extract more information from images of live cells than ever before. Next, we are likely to see the technology outfitted for higher-throughput systems—in drug-screening studies, for example. Also watch for label-free technology to become more prevalent in live-cell-based assays, as researchers strive to observe even more while interfering even less.