Human adherent cortical organoids in a multi-well format

In the growing diversity of human induced pluripotent stem cell (iPSC)-derived models of brain development, we present here a novel method that exhibits 3D cortical layer formation in a reproducible topography of minimal dimensions. The resulting adherent cortical organoids (ACOs) develop by self-organization after seeding frontal cortex-patterned iPSC-derived neural progenitor cells in 384-well plates during 8 weeks of differentiation. The organoids have stereotypical dimensions of 3 × 3 × 0.2 mm, contain multiple subtypes of neurons, astrocytes, and oligodendrocyte lineage cells, and are amenable to extended culture for at least 10 months. Longitudinal imaging revealed morphologically mature dendritic spines, axonal myelination, and robust neuronal activity. Moreover, ACOs compare favorably to existing free-floating brain organoid models on the basis of robust reproducibility in obtaining topographically standardized radial cortical structures and circumventing internal necrosis. Adherent human cortical organoids hold considerable potential for high-throughput drug discovery applications, neurotoxicological screening, and mechanistic pathophysiological studies of brain disorders.