After 10 day stationary culture, organoids in 96-well plate were transferred to ultra-low-attachment 6-well plate. Maximum 8 organoids were transferred into each well of the 6-well plate. To generated hMGEOs, ventral patterning media was used from day 10 to day 18, with media change every other day. The ventral patterning media contained DMEM-F12, 0.15% (w/v) Detrose, 100 μM β-Mercaptoethanol, 1% (v/v) N2 supplement, 2% B27 supplement without vitamin A, 100 ng/ml recombinant SHH, and 1 μM purmorphamine. To generate hCOs, neural differentiation media without vitamin A was used from day 10 to day 18. The media contained 1:1 mixture of DMEM-F12 media and Neurobasal media, supplemented with 0.5% (v/v) N2 supplement, 1% (v/v) B27 supplement without vitamin A, 1% (v/v) Glutamx, 0.5% (v/v) MEM-NEAA, 0.025% (v/v) human insulin solution, 50 μM β-Mercaptoethanol, and 1% (v/v) Penicillin/Streptomycin. Cell culture plate was kept on orbital shaker with speed of 80 rpm/min, and media was replenished every other day.
