Generation of human cerebral organoids

Cerebral organoids were generated as previously described (Lancaster & Knoblich, 2014). Briefly, mycoplasma‐free iPSCs were dissociated into single cells using StemPro Accutase Cell Dissociation Reagent (A1110501, Life Technologies) and plated in the concentration of 9,000 single iPSCs/well into low attachment 96‐well tissue culture plates in hES medium (DMEM/F12 GlutaMAX supplemented with 20% knockout serum replacement, 3% ES grade FBS, 1% non‐essential amino acids, 0.1 mM 2‐mercaptoethanol, 4 ng/ml bFGF, and 50 μM Rock inhibitor Y27632) for 6 days in order to form embryoid bodies (EBs). Rock inhibitors Y27632 and bFGF were removed on the 4^th day. On day 6, EBs were transferred into low attachment 24‐well plates in NIM medium (DMEM/F12GlutaMAX supplemented with 1:100 N2 supplement, 1% non‐essential amino acids, and 5 μg/ml heparin) and cultured for additional 6 days. On day 12, EBs were embedded in Matrigel drops and then they were transferred in 10‐cm tissue culture plates in NDM minus A medium (DMEM/F12 GlutaMAX and Neurobasal in ratio 1:1 supplemented with 1:100 N2 supplement 1:100 B27 without vitamin A, 0.5% non‐essential amino acids, insulin 2.5 μg/ml, 1:100 antibiotic–antimycotic, and 50 μM 2‐mercaptoethanol) in order to form organoids. 4 days after, Matrigel embedding cerebral organoids were transferred into an orbital shaker and cultured until electroporation in NDM plus A medium (DMEM/F12 GlutaMAX and Neurobasal in ratio 1:1 supplemented with 1:100 N2 supplement 1:100 B27 with vitamin A, 0.5% non‐essential amino acids, insulin 2.5 μg/ml, 1:100 antibiotic–antimycotic, and 50 μM 2‐mercaptoethanol). During the whole period of cerebral organoid generation, cells were kept at 37°C, 5% CO₂ and ambient oxygen level with medium changes every other day. After transferring the cerebral organoids onto the shaker, medium was changed twice per week.