One should also anticipate considerable variability among organoids within the same cell line. Due to the unpatterned nature of the protocol, the number and size of neuroepithelial structures can vary between individual organoids. Typically, one can expect at least two large (>200 µm in diameter by D24) neural rosettes per organoid. For this reason, longitudinal studies, such as the observation of change in organoid size upon infection, are particularly enlightening. Variability between batches may also occur, with the most likely cause for this being inaccurate cell counting or seeding. It is recommended to conduct multiple counts and to make sure the cell suspension is well mixed before seeding onto the ULA U-bottom 96-well plates.
