Functional And Mechanistic Neurotoxicity Profiling Using Human iPSC-Derived Neural Spheroid 3D Cultures

There is an increasing interest in using more complex, biologically relevant, and predictive cell-based assays for assay development and compound screening. StemoniX microBrain® 3D Assay Ready Platform is a high throughput 3D culture platform that more closely resembles the tissue development and constitution of native human brain tissue. In this platform, human iPSC-derived neuronal spheroids are composed of a physiologically relevant co-culture of functionally active cortical glutamatergic and GABAergic neurons and astrocytes. This balanced cellular mix allows the development of a neural network enriched in synapses, creating a highly functional neuronal circuitry. The neuronal cells in the microBrain 3D spheroids are physiologically active, with spontaneous synchronized, readily detectable calcium oscillations.

We used fast kinetic fluorescence imaging on the FLIPR® Tetra System to measure the patterns and frequencies of the Ca2+ oscillations of neuro-spheroids as monitored by changes in intracellular Ca2+ levels with calcium-sensitive dyes. A set of known neuromodulators was tested, including agonists and antagonists of NMDA, GABA and AMPA receptors; kainic acid, analgesic, and anti-epileptic drugs. Changes were observed as inhibitions or activations of the oscillation patterns, matching the expected effect of the correspondent neuromodulator. In addition, we tested a set of known neurotoxic compounds including selected pesticides and flame retardants and demonstrated sensitivity of the assay to the effects of compounds. Assay was optimized for HTS in 384-well plates and allows for the characterization of oscillation profiles in neural spheroid by using multi-parametric analysis with ScreenWorks Peak Pro Software. The automatically measured read-outs include the oscillation rate, peak frequency, peak width, amplitude, and waveform irregularities. In addition, the potential impact of treatment on cell viability and mitochondrial integrity was evaluated by high content imaging using ImageXpress® Micro Confocal High-Content Imaging System. We determined EC50s for the impact of different compounds on the Ca2+ oscillation rates or cell viabilities. In conclusion, we demonstrated that functional and morphological assays using 3D neuronal spheroids formed with human iPSC-derived cells can be used for evaluation of drug candidates and neurotoxicity assessment.

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