Fig. 1
Generation of A-3D neuronal cultures in 96-well plates. The top panel shows a schematic representation of the differentiation procedure. Neural precursor cells (NPCs) are seeded at a density of 2.5 × 105 cells/well on matrigel-treated optical active 96-well plates. Cells are differentiated for at least 4 weeks as described in the Methods section. During this period, NPCs self-assemble and organize into multiple layers. The “spheroidalization” of these multilayered cell aggregates is presumably prevented by the coating of culture wells with matrigel. Matrigel is removed before seeding the cells. During the differentiation process, cells migrate toward the center of the culture wells with a consequent increase in the thickness of the 3D cell aggregates. The images show confocal microscopy analysis of 3D cellular aggregates. Sections (145 μm × 145 μm × 30–55 μm) were generated and are shown in different orientations. a–c Staining for Nestin/TUJ1: a 3D rendering in an angled orientation; b an “en face” view of the 3D rendering; c viewing along the depth of the section. d–f Staining for chondroitin sulfate: d 3D rendering in an angled orientation; e image of one of the z stacks; f viewing along the depth of the section. g–i Staining for MAP2/GFAP. j 3D rendering in an angled orientation of the staining for Vimentin/MAP2. k Staining for CUX2 (viewing along the depth of the section). l Staining for VGLUT1. m,n 3D rendering in an angled orientation of the staining for CALBINDIN (m) and CTIP2 (n). o Staining for Tau. p Quantification of MAP2-positive and Vimentin-positive cells by high-content imaging. The data represent an average of three independent experiments. Error bars represent standard deviations. Cell nuclei are counterstained with TO-PRO-3 (TOPRO). TOPRO staining shows the presence of multiple cell layers. Scale bar = 20 μm in b, e, and o; = 50 μm in l