Differentiation of NPCs into Brain Organoids

NPCs were differentiated into brain organoids according to published protocols [50 (link), 51 (link)] with minor modifications. NPCs were dissociated with accutase (Sigma-Aldrich), briefly centrifuged, and resuspended in NPC medium. Cells were counted using the TC20 automated cell counter (Bio-Rad) and plated at a density of 9000 cells/well on BIOFLOAT™ 96-well plates (faCellitate, F202003). The plate was centrifuged at 220 × g for 5 min and placed in the incubator at 37 °C/5% CO₂. After 48 h, the medium was switched to patterning medium (N2B27 supplemented with 200 μM AA, 1 ng/mL BDNF (Prospec, CYT-207), 1 ng/mL GDNF (PeproTech, 450-10), and 1 µM SAG). After 2 days, the brain organoid media was refreshed with patterning medium. After 2 days, the medium was replaced with maturation medium (N2B27 supplemented with 200 μM AA, 2 ng/mL BDNF, 2 ng/mL GDNF, 1 ng/mL TGF-β3 (Prospec, CYT-368), 5 ng/mL ActivinA (Stem Cell Technologies, 78001), and 100 μM dbcAMP (Sigma-Aldrich, D0260). ActivinA concentration was lowered to 2 ng/mL for the subsequent media changes. The medium was changed every other day. On day 30, the medium was switched to N2B27 without supplements and refreshed every other day.