Gibco DMEM/F-12, HEPES

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	Culture in orbital shaker and change medium every 2-3 days Characterized brain organoids at day 45–75 of differentiation

Protocol tips
Culture in orbital shaker and change medium every 2-3 days Characterized brain organoids at day 45–75 of differentiation

Publication protocol

hiPSC-derived brain organoids were generated based on the protocol described by Lancaster et al.15 with modifications. Briefly, hiPSCs were generated from IMR-90 and AG14048 human fibroblasts, confirmed to be karyotypically normal and negative for mycoplasma contamination. On day 0 of organoid culture, hiPSCs were dissociated with EDTA, and seeded in suspension in a 6-well plate to form embryoid bodies in E8 medium with 5 μM ROCK inhibitor Y-27632. From day 1 to day 4, cells were cultured in E8 medium without ROCK inhibitor with daily medium change. On day 5, E8 medium was replaced by neural induction medium (NIM) containing DMEM-F12, 1 × N2 supplement, 1 × minimum essential medium NEAA (MEM-NEAA), and 2 μg/ml Heparin. On day 8, the spheres were embedded in 20%–25% Matrigel in NIM in a 6-well suspension plate and incubated at 37°C for 4 hr, followed by gentle addition of 2 mL of the NIM. On day 10-12, brain organoids were lifted and transferred to a new 6-well plate. NIM was changed daily from day 5 to day 15. On day 15, brain organoids were transferred to a T25 suspension culture flask and cultured in differentiation medium containing DMEM-F12, 1 × N2 supplement, 2.5 μg/ml Insulin, 1 × Glutamax, 0.5 × MEM-NEAA, 3.5 μl/L (V/V) 2-Mercaptoethanol, and 1 × B27 supplement on an Orbi-Shaker (Benchmark Scientific) at 50 rpm rotating speed. Medium was changed every 2-3 days. Organoids that exhibited similar size and passed the quality control criteria described by Lancaster et al.15,18 were used for the study. The criteria include clear embryoid body border, formation of organized neuroepithelium before embedding, formation of ventricle-like structure, and development of defined bud in Matrigel without premature differentiation.

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