Cerebral Organoid Differentiation Protocol

The second differentiation protocol was adapted from a protocol of cerebral organoid formation [18 (link)]: Fifty thousand cells per well were plated inside a u-shaped ultra-low attachment 96-well-plated (Thermo Fisher Scientific, Waltham, MA, USA) in neural progenitor medium. Cells aggregated into spheres for 7 days, were removed from the wells and embedded in groups of 5 inside 50 µL droplets of ice-cold Matrigel (Thermo Fisher Scientific, Waltham, MA, USA) on a sheet of sterilized parafilm (Bemis, Neenah, WI, USA). After polymerization for 30′ at 37 °C, the droplets were removed from the film, transferred into ultra-low attachment 6-well plates (Thermo Fisher Scientific, Waltham, MA, USA) and differentiation induced by differentiation medium for four weeks.