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There is an increasing interest in using three-dimensional (3D) cell structures for modeling tumors, organs, and tissue to accelerate translational research. We describe here a novel automated organoid assay system (the Pu·MA System) combined with microfluidic-based flowchips that can facilitate 3D cell-based assays. The flowchip is composed of sample wells, which contain organoids, connected to additional multiple wells that can hold various assay reagents. Organoids are positioned in a protected chamber in sample wells, and fluids are exchanged from side reservoirs using pressure-driven flow. Media exchange, sample staining, wash steps, and other processes can be performed without disruption to or loss of 3D sample. The bottom of the sample chamber is thin, optically clear plastic compatible with high-content imaging (HCI). The whole system can be kept in an incubator, allowing long-term cellular assays to be performed. We present two examples of use of the system for biological research. In the first example, cytotoxicity effects of anticancer drugs were evaluated on HeLa and HepG2 spheroids using HCI and vascular endothelial growth factor expression. In the second application, the flowchip system was used for the functional evaluation of Ca2+ oscillations in neurospheroids. Neurospheres were incubated with neuroactive compounds, and neuronal activity was assessed using Ca2+-sensitive dyes and fast kinetic fluorescence imaging. This novel assay system using microfluidics enables automation of 3D cell-based cultures that mimic in vivo conditions, performs multidosing protocols and multiple media exchanges, provides gentle handling of spheroids and organoids, and allows a wide range of assay detection modalities.

All eukaryotic cells tightly control cellular pH. Proper control of cytoplasmic pH is essential for normal metabolism and cell growth, and acidification of organelles such as the lysosome, endosome, and Golgi apparatus is essential for protein sorting and degradation, ion homeostasis, and signal transduction. The vacuolar ATPase (V-ATPase) is one of the central players in pH control. All eukaryotic cells have V-ATPases of remarkably similar structure, and loss of V-ATPase function is lethal at early stages of development in higher eukaryotes and conditionally lethal in fungi.

Neurological disorders affect millions of people worldwide and appear to be on the rise. Whereas the reason for this increase remains unknown, environmental factors are a suspected contributor. Hence, there is an urgent need to develop more complex, biologically relevant, and predictive in vitro assays to screen larger sets of compounds with the potential for neurotoxicity. Here, we employed a human induced pluripotent stem cell (iPSC)-based 3D neural platform composed of mature cortical neurons and astrocytes as a model for this purpose. The iPSC-derived human 3D cortical neuron/astrocyte co-cultures (3D neural cultures) present spontaneous synchronized, readily detectable calcium oscillations. This advanced neural platform was optimized for high-throughput screening in 384-well plates and displays highly consistent, functional performance across different wells and plates. Characterization of oscillation profiles in 3D neural cultures was performed through multi-parametric analysis that included the calcium oscillation rate and peak width, amplitude, and waveform irregularities. Cellular and mitochondrial toxicity were assessed by high-content imaging. For assay characterization, we used a set of neuromodulators with known mechanisms of action. We then explored the neurotoxic profile of a library of 87 compounds that included pharmaceutical drugs, pesticides, flame retardants, and other chemicals. Our results demonstrated that 57% of the tested compounds exhibited effects in the assay. The compounds were then ranked according to their effective concentrations based on in vitro activity. Our results show that a human iPSC-derived 3D neural culture assay platform is a promising biologically relevant tool to assess the neurotoxic potential of drugs and environmental toxicants.

This study set out to develop a high-throughput multiplexed assay using iPSC-derived skeletal myoblasts that can be used as a first-pass screen to assess the potential for chemicals to affect skeletal muscle. We found that cytotoxicity and cytoskeletal integrity are most useful and reproducible assays for the skeletal myoblasts when evaluating overall cellular health or gauging disruptions in actin polymerization following 24 h of exposure. Both assays are based on high-content imaging and quantitative image processing to derive quantitative phenotypes. Both assays showed good to excellent assay robustness and reproducibility measured by interplate and interday replicability, coefficients of variation of negative controls, and Z′-factors for positive control chemicals. Concentration response assessment of muscle-related toxicants showed specificity of the observed effects compared to the general cytotoxicity. Overall, this study establishes a high-throughput multiplexed assay using skeletal myoblasts that may be used for screening and prioritization of chemicals for more complex tissue chip-based and in vivo evaluation.

Development of more complex, biologically relevant, and predictive cell-based assays for compound screening is a major challenge in drug discovery. The focus of this study was to establish high-throughput compatible three-dimensional (3D) cardiotoxicity assays using human induced pluripotent stem cell-derived cardiomyocytes. Using both high-content imaging and fast kinetic fluorescence imaging, the impact of various compounds on the beating rates and patterns of cardiac spheroids was monitored by changes in intracellular Ca2+ levels with calcium-sensitive dyes. Advanced image analysis methods were implemented to provide multiparametric characterization of the Ca2+ oscillation patterns. In addition, we used confocal imaging and 3D analysis methods to characterize compound effects on the morphology of 3D spheroids. This phenotypic assay allows for the characterization of parameters such as beating frequency, amplitude, peak width, rise and decay times, as well as cell viability and morphological characteristics. A set of 22 compounds, including a number of known cardioactive and cardiotoxic drugs, was assayed at different time points, and the calculated EC50 values for compound effects were compared between 3D and two-dimensional (2D) model systems. A significant concordance in the phenotypes was observed for compound effects between the two models, but essential differences in the concentration responses and time dependencies of the compound-induced effects were observed. Together, these results indicate that 3D cardiac spheroids constitute a functionally distinct biological model system from traditional flat 2D cultures.In conclusion, we have demonstrated that phenotypic assays using 3D model systems are enabled for screening and suitable for cardiotoxicity assessment in vitro.

Cell models are becoming more complex to better mimic the in vivo environment and provide greater predictivity for compound efficacy and toxicity. There is an increasing interest in exploring the use of three-dimensional (3D) spheroids for modeling developmental and tissue biology with the goal of accelerating translational research in these areas. Accordingly, the development of high-throughput quantitative assays using 3D cultures is an active area of investigation. In this study, we have developed and optimized methods for the formation of 3D liver spheroids derived from human iPS cells and used those for toxicity assessment. We used confocal imaging and 3D image analysis to characterize cellular information from a 3D matrix to enable a multi-parametric comparison of different spheroid phenotypes. The assay enables characterization of compound toxicities by spheroid size (volume) and shape, cell number and spatial distribution, nuclear characterization, number and distribution of cells expressing viability, apoptosis, mitochondrial potential, and viability marker intensities. In addition, changes in the content of live, dead, and apoptotic cells as a consequence of compound exposure were characterized. We tested 48 compounds and compared induced pluripotent stem cell (iPSC)-derived hepatocytes and HepG2 cells in both two-dimensional (2D) and 3D cultures. We observed significant differences in the pharmacological effects of compounds across the two cell types and between the different culture conditions. Our results indicate that a phenotypic assay using 3D model systems formed with human iPSC-derived hepatocytes is suitable for high-throughput screening and can be used for hepatotoxicity assessment in vitro.

3D organotypic culture models such as organoids and multicellular tumor spheroids (MCTS) are becoming more widely used for drug discovery and toxicology screening. As a result, 3D culture technologies adapted for high-throughput screening formats are prevalent. While a multitude of assays have been reported and validated for high-throughput imaging (HTI) and high-content screening (HCS) for novel drug discovery and toxicology, limited HTI/HCS with large compound libraries have been reported. Nonetheless, 3D HTI instrumentation technology is advancing and this technology is now on the verge of allowing for 3D HCS of thousands of samples. This review focuses on the state-of-the-art high-throughput imaging systems, including hardware and software, and recent literature examples of 3D organotypic culture models employing this technology for drug discovery and toxicology screening.