Gibco™Neurobasal™ Medium

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Replace medium (2 ml) every 72 h for the first 30 d and then increased to 3 ml for the duration of culture Replace the plates every 30 d to prevent buildup of debris

Protocol tips
Replace medium (2 ml) every 72 h for the first 30 d and then increased to 3 ml for the duration of culture Replace the plates every 30 d to prevent buildup of debris

Publication protocol

Generation of cerebral organoids: ESCs were singularized using TrypLe Select (#12563011; Life Technologies) and resuspended at 80,000 cells/ml in EB media (DMEM/F12, 20% KSR, 2% MEM-NEAA, 55 μM β-mercaptoethanol) with 4 ng/ml bFGF and 50 μM Y-27632 (Y27) (#S1049; Selleck Chem). The cells (12,000 cells/well) were plated in 96-well V-bottom nonbinding plates (#651970; Greiner Bio-One), and on Day 2, fresh EB medium containing 2 ng/ml bFGF was added. On Day 5, healthy EBs with a diameter of 425–475 μm were transferred to 24-well ultralow attachment plates (#CLS3473; Corning) in 500 μl neural induction medium (DMEM/F12, 1% N2, 1% MEM-NEAA, 1% GlutaMax, and 1 μg/ml heparin [#H3393; Sigma-Aldrich]), and 48 h later, an additional 500 μl of neural induction medium was added. EBs were transferred to pre-warmed four-well tissue culture plates, excess medium was aspirated, and freshly thawed growth factor reduced Matrigel (30 μl) was added on top of EBs. Plates were transferred to a 37°C CO2 incubator for 10 min to allow the Matrigel to polymerize, and then 500 μl of cerebral organoid differentiation media without vitamin A (CDM − Vit A; 48% DMEM/F12, 48% neurobasal [#21103049; Life Technologies], 0.5% MEM-NEAA, 1% GlutaMax, 0.5% N2, 1% B27 without vitamin A [#12587001; Life Technologies], 2.5 μM insulin, and 192.5 μM β-mercaptoethanol) was added to each well. On Day 11, the medium was replaced, and on Day 13, spheroids containing ring-like structures were extracted using a Scalpel (#1000044; Thermo Fisher Scientific), ensuring that excess Matrigel is removed. Organoids (maximum of three per well) were transferred to a six-well tissue culture plate containing 3 ml of CDM + Vit A (CDM with 1% B27 containing Vit A) and then placed on an orbital shaker (#88881101; Thermo Fisher Scientific) at 90 rpm (9.5 mm radius) in a CO2 incubator for the duration of culturing. The medium (2 ml) was replaced every 72 h for the first 30 d and then increased to 3 ml for the duration of culture. The plates were replaced every 30 d to prevent buildup of debris. Spheroid formation efficiency was tested using nonbinding V-bottom plates (#651970; Greiner Bio-One), Aggrewell800 plates (#38421; StemCell Tech) coated with Anti-Adherence Rinsing Solution (#07010; StemCell Tech), U-bottom non-treated polystyrene plates (#168136; Thermo Fisher Scientific), and U-bottom Ultra-Low Attachment plates (#4515; Corning).

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Manufacturer protocol

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