To generate the fusion organoids, two VOr EBs and one BOr EB were collected and then embedded
together into one Matrigel droplet (25 μl) on day 12. The two VOr EBs were put on two sides of the BOr EB, and pipette tips could be used to adjust the shape and site of the three EBs and then cultured with VEGF-containing (20 ng/ml) neural differentiation medium (50% [v/v] DMEM/F12 and
50% [v/v] Neurobasal medium [Life/Invitrogen] containing 0.5% [v/v] N2 supplement, 0.5% [v/v] B27
supplement without vitamin A [Life/Invitrogen], 3.5 μl/l β-mercaptoethanol, 250 μl/l insulin [SigmaAldrich], 1% [v/v] GlutaMAX, and 0.5% [v/v] MEM-NEAA, 1% [v/v] Antibiotic-Antimycotic [Gibco]).
After 4 days, the differentiation medium was replaced by VEGF-containing (20 ng/ml) maturation medium (50% [v/v] DMEM/
F12 and 50% [v/v] Neurobasal medium containing 0.5% [v/v] N2 supplement, 0.5% [v/v] B27 supplement [Life/Invitrogen], 3.5 μl/l β-mercaptoethanol, 250 μl/l insulin, 1% [v/v] GlutaMAX, and 0.5% [v/v]
MEM-NEAA, 1% [v/v] Antibiotic-Antimycotic). Then, the organoids were transferred into a shaker in the 5% CO2 incubator at 37°C for maturation, and the medium was renewed every 3–4 days.