Sr. Scientist, Sr. Manager of Assay Development Molecular Devices, LLC San Jose, California, United States
Abstract: Growing Neural 3D organoids from human induced pluripotent stem cells (iPSC) is a rapidly developing technology with great potential for understanding brain development, neuronal diseases, as well as impact of different genetic backgrounds. However, the formation of neural organoids is a complex and lengthy process, making it difficult for compound screening. Here we demonstrate automated protocol for development of forebrain neural organoids using CellXpress.ai cell culture system and Dorsal Forebrain organoid differentiation media. The CellXpress.ai has liquid handling, imager, automated incubator as well as process controlling scheduling software. The device enables automation of most repetitive tasks, including culture and expansion of iPSC, formation of organoids in AggreWell plates, then transfer and culture 3D organoids in 24well low attachment plates with periodic media exchanges, agitation, and monitoring organoid development by imaging. After 50 days of culture and maturation of 3D organoids functional characterization of spontaneous neural activity was measured by recording and analysis of calcium oscillations. Calcium oscillations were recorded after addition of calcium sensitive dye on a FLIPR instrument that measured fast kinetic changes in calcium signal. Oscillation patterns were analyzed by software for multiple parameters including peak count, amplitude, and peak width. Organoids demonstrated spontaneous calcium oscillation activity, with consistent rate of oscillations. Morphological characterization of 3D organoids was done by imaging. Organoids diameters ranged from 1800-2000 µm, and the expression of neural markers including TUJ1 and GAFP was detected with fluorescently labeled antibodies. For pharmacological characterization, several compounds were used to show the appropriate functional responses. AMPA and 4-AP addition resulted in dose-dependent increase of frequency of calcium oscillations, while GABA and caused decrease in oscillation frequencies. Taken together, this biological system of iPSC-derived 3D neural organoids paired with process automation and detailed analysis of calcium oscillations demonstrates a promising tool for compound testing.
