FIG 4.

Generation of brain organoids in Millicell-96 cell culture insert plates. (A) Schematic representation of the differentiation procedure. NPCs were seeded at a density of 3 × 10⁵ cells/well on 96-transwell plates. Cells were differentiated for 3 to 4 weeks, as described in the Materials and Methods section. (B) During this period, NPCs self-assembled and organized into multiple layers, forming quasispheroidal structures. (C) These 3D structures were then collected, transferred individually into low-attachment 24-well plates (where, over the course of hours, they assumed a roughly spherical shape), and cultured for an extended period of time. (D) The average size of the organoids after 8 weeks of NPC differentiation is depicted. (E) Hematoxylin and eosin staining of a 10-week-old organoid. (F) Wide-field micrograph of a formalin-fixed, paraffin-embedded section coimmunostained with Tuj1 and nestin. Nuclei were counterstained with Hoechst 33342. Bars, 500 mm (B) and 50 mm (F).