Once the cells are seeded and spun down in a ULA U-bottom 96-well plate (Figure 2A), they will appear as a flat, dense sheet of cells on the bottom of each well. The cells will slowly aggregate over the next two days. It is best not to disturb the plate during these 48 hours, as mechanical forces may disrupt the loosely-formed aggregate. For the first 10 days, the organoid will remain mostly spherical, and will grow from ~300 µm to 600-800 µm (Figure 2B). More observable structures begin to appear in the organoid between day 10 and day 20(Figure 2C-E). By day 24, researchers should be able to observe rosette-like structures via brightfield microscopy (Figure 2F). While continuing the culture, these neuroepithelial structures may increase in quantity and size over several days (Figure 2G). During feeds, the researcher will notice considerable amounts of debris collecting on the bottom of each well, particularly during the first 2 weeks of culture. The dispersion technique described in step 2.2 removes most of this cellular debris to prevent the apoptotic debris from accumulating over
