Category: Assay Development and Screening
The absence of physiologically relevant in vitro models of the nervous system is an important limitation in understanding mechanisms of neurological diseases and drug development. This has generated an increasing interest in using three-dimensional (3D) cultures for assay development applicable for neurodegenerative diseases and neurotoxicology screens. The goal of the present study was to develop a model for 3D neurite outgrowth assessment using iPSC-derived neurons in the microfluidic, high-throughput OrganoPlate platform.
The OrganoPlate was developed as an organ-on-a-chip platform allowing the formation of three-dimensional (3D), microfluidic-based, long-term cultures of live cells suitable for screening. Neuronal cultures were treated for five days with several compounds including methyl mercury and other selected chemicals that are known to inhibit neurite outgrowth. To assess the neuronal viability and complexity of networks, cells were stained using a combination of three dyes: Calcein AM, MitoTracker Orange, and Hoechst nuclear dye. We optimized the methods for assessing morphology and viability of neurons in 3D matrix using automated confocal imaging and analysis. Phenotypic readouts allowed quantitative characterization of the extent and complexity of the neural networks in 3D. Disintegrations of neuronal connections were visible in a dose-dependent manner after treatment with neurotoxic compounds. A series of confocal images were automatically acquired at different planes separated by 3-10 µm, covering approximately 150-300 µm in depth. Images were analyzed using our 3D analysis module in MetaXpress analysis software which allows us to define and characterize the neurite outgrowth, cell bodies, and nuclei. Objects were identified within each plane and then connected and measured in 3D space. The 3D analysis readouts included the number of neurites (fibers), volume, or the total volume of fibers, numbers of processes, or branching points. In addition, we applied 3D cell scoring analysis to count and characterize live cells, cells with intact mitochondria, as well as cell volume or fluorescence intensities for different markers.
Phenotypic readouts allowed quantitative characterization of the extent and complexity of the neural networks in 3D. Multiple measurements were used for defining effective concentrations for neurotoxicity. In addition, we evaluated assay reproducibility and tested a set of known neurotoxic compounds. The proposed method is extensively applicable for compound screening for prediction of neurotoxicity in a high-throughput manner.
Oksana Sirenko– Sr. Scientist, Molecular Devices LLC, Sunnyvale, California