A trending need in pharmaceutical and academic research environments is an improved, innovative and more efficient process for more relevant, predictive and functional cell-based assays in the drug discovery process. Using assays that need a label in traditional drug discovery has led to a limited view of drug effects and interferences from compounds in assays, resulting in false positives, false negatives and data of limited breadth. Also, when using labeled technologies, engineered cell lines are sometimes required, which can deviate from a true predictive model of cell biology.
More recently, label-free technology has been demonstrated to be a robust, highly sensitive, automatable tool used to measure live, native cells in real time. Furthermore, there is no need for dyes, engineered cells, tags or special reagents. The simple requirements for label-free assays lower the need for resources, simplify assay technique, reduce artifacts and enable the use of native cell types, including stem and primary cells. Most importantly, label-free technology allows for measuring the integrative phenotypic response resulting from multiple cellular pathway interactions. This enables scientists to observe the entire biological model instead of one biomarker of interest. Accumulating examples demonstrate the use of label-free screening assays to yield a higher rate of success in prioritizing and advancing lead compounds in drug discovery research environments. Due to the holistic nature of the data generated, label-free assays enable the selection of better, more relevant, surrogate cellular models. The data generated in this whole cell phenotypic assay is more physiologically relevant, and more accurately predict target biology for widely used disease models for neurodegeneration, cancer, diabetes and liver toxicity. Label-free assays, being phenotypic by nature, have the potential to generate leads targeting new molecular mechanisms of action (MMOAs), and hence truly innovate medicine.
As a goal to aid drug discovery and research, PerkinElmer (Waltham, Mass.) has incorporated Corning Life Sciences’ (Kennebunk, Maine) Epic optical label-free technology into their EnSpire Multimode Plate Reader, offering an orthogonal, nonintrusive and phenotypic approach to studying all cell types. Optical label-free technology measures changes in light refraction resulting from dynamic mass redistribution (DMR) within the cell. This redistribution occurs in response to receptor activation, pathway activation, or deactivation in the cell’s monolayer. DMR is the result of a series of biochemical signaling interactions within a cell and includes mass movements of mostly proteins as shown in Figure 1. Mass relocation as a cellular response to a stimulus is involved in most biological events and can be detected with label-free technology. The response is indicated by a wavelength change in the reflected light.
PerkinElmer label-free technology can be used in multiwell plate-based formats to noninvasively identify and characterize multiple receptor pathways in native and stem cell lines, as well as in recombinant cells if desired. By successfully monitoring the ligand-induced DMR in living cells, PerkinElmer has demonstrated that label-free technology is a sensitive and versatile tool for physiologically-relevant research, allowing for a broad range of applications including ligand binding, receptor activation, receptor inhibition, signaling propagation, intracellular recruitment, chemotaxis, cytotoxicity, ion channels, viral infection, endocytosis and cell morphological changes. The experimental design and assay workflow of label-free assays is very straightforward and special techniques are not required as shown in Figure 2.
Combining label-free and iPS cell technologies for neurodisease research
It is predicted that neurodegenerative diseases are destined to become the latest health crusade over the next several decades, therefore an improved understanding of neurotransmitters in the brain and knowledge of the effects of drugs on the corresponding receptors comprise one of the largest research efforts in neuroscience. Scientists hope that more relevant cell models and novel assays will help them become more knowledgeable around the circuits responsible for disorders such as Huntington’s disease, amyotrophic lateral sclerosis, Parkinson’s disease and Alzheimer’s disease. The number of people affected by these diseases is projected to grow significantly as the population ages. Currently, approved treatments for most of these neurodegenerative diseases do not modify the course of the disease, but instead only offer temporary relief of some symptoms and have only limited effectiveness. Additionally, existing cell sources for disease modeling, drug discovery and toxicity testing are limited by availability, functionality, reproducibility and translatability. All these factors combined with the complexity of the nervous system make it difficult to understand the underlying processes of neurodegeneration. Simpler whole-cell assay formats combined with better disease modeling would significantly advance the understanding of these illnesses, ultimately leading to disease-modifying drugs.
functions and characteristics of normal human neurons, including the endogenous expression of various ion channels and G protein-coupled receptors (GPCRs), providing a unique in vitro system for preclinical drug discovery, neurotoxicity testing, predictive disease modeling and basic cellular research.In addressing the increased demand for simpler, more physiologically-relevant assay platforms in drug discovery research, PerkinElmer has successfully bridged the gap between in vitro and in vivo pharmacology by combining label-free technology with induced pluripotent stem (iPS) cells to study and interrogate relevant neurodegenerative disease models. In collaboration with Cellular Dynamics International’s (Madison, Wisc.) iCell technology, methods have been successfully developed utilizing the PerkinElmer EnSpire Multimode Plate Reader with Corning Epic label-free technology, and the JANUS Automated Workstation, to noninvasively and phenotypically analyze iPSC-derived neurons. iPS cell technology revolutionizes life-science research and personalized medicine by enabling disease modeling in a biologically relevant system, while dually serving as a powerful tool to better understand and target human disease. iCell Neurons are derived from human induced pluripotent stem (iPS) cells and display physiological
By monitoring the ligand-induced DMR in iCell Neurons, the EnSpire label-free platform detects the whole cell’s response.
Interrogating GABAA receptor
As an example of how phenotypic assays apply to neurodegenerative disease models, we present the application of label-free technology to interrogate the endogenously expressed GABAA receptor in iCell Neurons, which is a ligand-gated ion channel. The first step in the assay development process is to determine the optimal signal required for the label-free assay with this particular cell type. Therefore iCell Neurons were seeded at three different densities and treated with a dose response of GABA on Day 5 post-thaw. The label-free response was monitored over 60 minutes on the EnSpire multimode plate reader, and the peak DMR values were used to generate concentration-response curves (and EC50 values) for each density. As shown in Figure 3, a robust response was observed at each density—with 20,000 cells per well providing the maximum signal. However, using lower cell numbers is acceptable and may provide an additional cost savings benefit when running this assay.
The next logical step would be to interrogate the receptor of interest for functionality. In order to determine whether the EnSpire label-free can be used to detect a GABAA-specific response, the chemical neurotransmitter gamma aminobutyric acid (GABA) was added to iCell Neurons and the DMR responses were monitored. As shown in Figure 4, activation of the receptor with GABA was dose-dependent and saturable with an EC50 value around 1 ìM (n=4). This is in good agreement with literature values using other technologies such as ion flux assays or whole cell patch-clamp recordings and the assay is shown to be highly reproducible. In contrast, the antagonist GABAzine was able to inhibit the cellular response in the presence of GABA, showing that this system is a good model for studying the GABAA receptor and related pathways.
Modulation of endogenous receptor activity, such as for the GABAA ion channel receptor, requires that assay conditions be highly sensitive and robust. Figure 5 demonstrates the high quality of the GABAA assays on the EnSpire label-free instrument. The Z’ factor was determined by adding a stimulating dose of GABA (100 μM) in Hank’s Balanced Salt
Solution (HBSS) assay buffer to iCell Neurons at an optimized cell density of 15,000 cells per well and measuring the peak DMR response over 60 minutes. Using 45 wells for maximum and 45 wells for minimum signal in the assay, the Z’ factor was calculated and was shown to exceed 0.6. These data illustrate that the EnSpire label-free system can robustly perform stem cell screening assays, which proves to overcome a big hurdle in the screening world.
The combination of a novel phenotypic screening method with iPS cell-derived neurons, with the added utility of automated liquid handling to ensure robust and reproducible cellular assays, has been demonstrated. PerkinElmer’s novel label-free utilization sets the foundation for downstream interrogation of this native cell model for these neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease. Furthermore, this simplified label-free assay development with iPS cell-derived neurons can serve as an example for other stem cell disease models of interest.
Increased interrogation and MMOA elucidation
Label-free has been proven to be pathway unbiased, therefore the data is most valuable when used in conjunction with orthogonal labeled assays, which can be read using the other functionalities of the same Enspire reader. It is predicted that the trend in label-free will overlap with and evolve towards image-based, high-content models, leading to increased interrogation and MMOA elucidation in drug discovery. By joining label-free with iPS cell technology, a powerful model has been developed to include more physiologically relevant, predictive and innovative cell-based assays, enabling a revolution in drug discovery and personalized medicine.
Filed Under: Drug Discovery
