Protocol for the generation of three-dimensional micropatterned neuroepithelial tissues using hPSCs, bioprinting, and matrix scaffolds

Micropatterning technology that spatially guides the self-assembly of human pluripotent stem cells (hPSCs) into neural tissues offers enhanced fidelity to investigate early neurodevelopment and disease mechanisms. Here, we present a protocol to produce micropatterned and scaffolded neuroepithelial tissues (scNETs) from hPSCs, leveraging three-dimensional (3D) bioprinting to deposit extracellular matrix (ECM) droplets with defined geometries. We describe steps for bioprinting of ECM micropatterns, hPSC culture and seeding, and the subsequent neural induction process to form scNETs within 5 days. For complete details on the use and execution of this protocol, please refer to Imani Farahani et al.¹.