Male or female rats

Spheroid preparation proceeded as described in a previous study (Dingle et al., 2015). In brief, cortex was isolated from male or female rats (Charles River) at postnatal Day 0-2, diced, and enzymatically digested in 2 mg/mL solution of papain (BrainBits, LLC) in Hibernate A sans Ca 2+ (BrainBits, LLC) for 30 minutes at 30°C. The papain solution was removed, and cells were mechanically separated with a fire-polished Pasteur pipette in Hibernate A buffer supplemented with 1 x B27 (Invitrogen) and 0.5 mM GlutaMAX (Invitrogen). The cell solution was then centrifuged and resuspended in cortical media consisting of Neurobasal A (Invitrogen) supplemented with B27, 0.5 mM GlutaMAX, and Penicillin-Streptomycin (Invitrogen). The cell suspension was washed again by centrifuging and resuspending the pellet in cortical media. Cortical cells were placed in 2% agarose microarrays created by 400 µ m diameter round silicon peg molds (Microtissues, Inc) at a density of 4-8,000 cells/microwell. The cell suspension was allowed to settle in the microwells for 30-40 minutes, after which 1 mL of cortical media was added to each well of a 24-well plate. Spheroids were incubated at 37º C in 5% CO 2 , and the cortical media was changed every 2-3 days.