Protocol for minimizing necrotic core formation in iPSC-derived cortical organoids.

Cortical organoids (COs) derived from human induced pluripotent stem cells (hiPSCs) provide a unique opportunity to model human neurodevelopment in vitro. Here, we present a protocol to prevent necrotic core formation without requiring organoid slicing. We describe steps for hiPSC culturing, preparation of single-cell suspension, seeding, and embryoid body (EB) and neuroectoderm formation. We then detail procedures for CO formation and maintenance without necrotic core formation. This protocol sustains long-term viability and preserves structural integrity by enhancing oxygen and nutrient diffusion.