DISSORG: A HIGH-THROUGHPUT 2D CORTICAL ORGANOID-DERIVED NEURONAL AND GLIAL CO-CULTURE PLATFORM THAT IS CAPABLE OF SYNAPTIC PLASTICITY

Human induced pluripotent stem cells (hiPSCs) are increasingly used to bridge the translational gap between basic research and clinical applications. Brain organoids have mature, diverse cell types, but are limited in throughput and experimental accessibility and often exhibit restricted maturation due to necrotic core formation. We therefore developed a high-throughput, long-term functional hiPSC-derived neuron–astrocyte co-culture system using dissociated organoid (DissOrg) cultures. We dissociated hiPSC-derived cortical organoids (DIV70+) from two donor cell lines. Immunostaining confirmed the presence of mature neurons, interneurons, astrocytes and synapses. Calcium imaging detected spontaneous activity in neurons and astrocytes, with neuronal calcium signals enhanced by NMDA and gabazine, and astrocytic responses increased by ATP and suppressed by CPA. DissOrg cultures exhibited a progressive increase in mean firing rate and network bursts over 7 weeks and long-term potentiation (LTP) for up to 3 hours following chemical LTP in high-density multi-electrode array (HD-MEA) recordings. Transcriptomic profiling is in progress. As cortical organoids lack microglia, we supplemented DissOrg cultures with hiPSC-derived ioMicroglia (bit.bio), to model neuroimmune and inflammatory processes. ioMicroglia successfully integrated into the neuron-astrocyte network. We also characterized DissOrg cultures from Alzheimer’s patient lines (e.g., PSEN1) and found aberrant astrocyte morphology compared to donor cell lines. Overall, DissOrg cultures offer a humanized, scalable in vitro platform consisting of mature, functional neurons and astrocytes that can be supplemented with additional cell types like microglia or endothelial cells. This system provides a versatile approach for modelling healthy and diseased brain physiology and offers broad potential for translational neuroscience research.