A system for high-throughput axonal imaging of induced pluripotent stem cell-derived human i3Neurons

Neurons have long, thin axons and branched dendritic processes which rely on an extensive microtubule network that functions as a cellular scaffold and substrate for cargo transport. Microtubule defects are a defining pathological feature of neurological disorders. The highly arborized, long, polarized neuronal processes pose challenges for imaging-based assays. Available methods use either dispersed cultures, which are inefficient for compartment-specific analyses, or microfluidic chambers, which allow clear separation of somatodendritic and axonal compartments but are expensive and difficult to maintain. Here, we introduce an "i3Neurosphere" culture model of induced pluripotent stem cell (iPSC)-derived human cortical i3Neurons that enables high-throughput imaging of hundreds of axons without specialized equipment. We characterize neurite outgrowth, polarization, microtubule dynamics, and motility of diverse cargo, providing a reference for future work on microtubule processes in this system. The high-throughput compartment-specific imaging we present, combined with facile genetic engineering in i3Neurons provides a powerful tool to study human neurons. SIGNIFICANCE STATEMENTHuman neurons are difficult to study due to limited access to tissue and technical challenges in existing in vitro models of axonal transport. We developed i3Neurospheres, a simple and scalable 3D culture system of human iPSC-derived neurons that enables high-throughput imaging of axonal outgrowth, microtubule dynamics, and intracellular transport. This platform provides an accessible, reproducible method for investigating neuronal function and disease mechanisms, offering broad utility for neuroscience research and preclinical drug screening.