Figure 1

Overview of the human cerebral organoid generation protocol. (A) Human pluripotent stem cells are expanded in traditional 2D culture, dissociated, aggregated into microwells, and matured into 3D organoid cultures using defined media conditions to promote cerebral cortex tissue differentiation. In this study, on day 12 post-aggregation, organoids were either kept in suspension and maintained manually (black arrow) or transferred to individual wells of a microfluidic chip and maintained in automation (blue arrow). (B) Images of cerebral organoid cultures. Bright-field images at low (left) and high (center) magnification under standard culture conditions show organoid morphology and heterogeneity. Immunofluorescence stains on week 5 for PAX6 (green, radial glia progenitor cells), CTIP2 (BCL11B) (magenta, excitatory projection neurons), ZO-1 (TJP1) (white, tight junction proteins on radial glia endfeet, apical surface of the neural tube), show characteristic ventricular zone-like rosette structures with radial glia surrounded by neurons. Nuclei stained with DAPI (blue). (C) Image of the PDMS microfluidic chip. The custom cell culture chip, modeled after a standard 24-well plate, houses organoids for automated experiments.