To generate cerebral organoids, PGP1 iPSCs were grown to ~80% confluency and, on day 0, were lifted from Matrigel-coated T225 flasks by incubation with gentle cell dissociation reagent for 12 min. The flasks were rinsed with DMEM/F12 containing Hepes and centrifuged for 5 min at 220g. Next, the cells were resuspended in EBCM and 10 μM ROCKi and seeded into microwell arrays, prepared as described above, at a density of approximately 500 cells per microwell, and compacted by centrifugation at 100g for 3 min. The media was replaced on day 1 with fresh EBCM without ROCKi. On day 2, the EBs were measured to be approximately 200 μm in diameter, and the media was replaced with neural induction medium (NIM) composed of DMEM/F12 with GlutaMAX, 1:1000 of heparin (10 mg/ml; Sigma-Aldrich), 1:100 N-2 supplement (Thermo Fisher Scientific), and 1× minimum essential medium nonessential amino acids (MEM-NEAA) (Thermo Fisher Scientific) with 1:2000 of 10 mM SB431542 in dimethyl sulfoxide (DMSO) (SB, EMD Millipore) and 1:2000 of 0.2 mM LDN 193189 in DMSO (LDN, EMD Millipore). Aliquots of SB and LDN were stored at −20°C and added to the other media components immediately before adding NIM to cells. On day 3, the OBBs were removed from the microwells by pipetting up and down with DMEM/F12 containing 15 mM Hepes buffer, and the aggregates were transferred to a 50-ml centrifuge tube. The OBBs were allowed to settle under gravity, the supernatant was aspirated, and then, the OBBs were resuspended in 6 ml of NIM per two microwell arrays that were harvested. Next, they were seeded into untreated T25 flasks, and 6 ml of NIM was mixed with the OBBs per flask. After harvesting, an additional 5 ml of NIM was pipetted into each flask. The flasks were transferred to an orbital shaker (VWR Shaker, 1000 Standard) that was set to 60 rpm and placed in an incubator. The media was replaced on days 5 and 7 by placing the flasks on a custom-built sedimentation rack that held the flask at 45° such that cells settle into a single corner of the T25 flask. Once settled, the supernatant was aspirated and replaced with 6 ml of fresh NIM. On day 8, the medium was replaced with neural differentiation medium (NDM) composed of a 1:1 mix of DMEM/F12 with Neurobasal medium (Thermo Fisher Scientific), 1:200 N-2 supplement (Thermo Fisher Scientific), 1:100 B27 without vitamin A (Thermo Fisher Scientific), 1:4000 insulin solution (Sigma-Aldrich), 1:100 GlutaMAX, and 1:200 MEM-NEAA. A 1000× solution of β-mercaptoethanol (1:1000; Thermo Fisher Scientific) was added to NDM immediately before adding the media to the OBBs. Thereafter, the media was replaced every other day, and the cerebral organoids were harvested for SWIFT vascularization on day 21.

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