3D Cell-culture preparation

Hippocampal neuronal cultures were generated from E17 to E18 embryos by building upon standard cell-culture techniques, as follows. Pregnant mice were anesthetized under isoflurane, and euthanized by cervical dislocation. Hippocampus was dissected in Hibernate E (Gibco) iced cold media, and incubated in 0.25% Trypsin-EDTA (Gibco) at 37 °C for 30 min., followed by 5 min DNAse I (1 μg/ ml; Sigma) incubation at room temperature. Mechanical dissociation of dissected hippocampus was performed by repeated pipetting with a fire-polished glass Pasteur pipet until a homogenous cell suspension was obtained. Note that, for preparing MoNNets from CD1 mice, the dissociated cells from all pups in a given litter were mixed, whereas for MoNNets from Setd1a+/, Df(16)A^+/− and their WT siblings, cells from each embryo were processed separately to generate MoNNets. Cell viability was determined by Trypan Blue exclusion assay. The cell solution was then centrifuged at 150g for 10 min, and the supernatant was removed. The resulting cell pellet was resuspended in the culture media containing Neurobasal media, 2% B27, 0.5 mM Glutamate and 1% Penicillin/Streptomycin (Gibco). Cells were infected with AAV1.Syn.GCaMP6f.WPRE.SV40 virus (⁶³; Pennsylvania Vector Core, Cat#:AV-1-PV2822) for neuronal GCaMP6f expression. The viral infection at single-cell suspension stage, just before seeding, allowed for infection of most cells. It is well documented that following similar procedures, AAV1 is transduced in over 94.1% of all cells with approximately 80% of them being neurons⁶⁴. For single isolated spheroid cultures, 2% agarose 96 wells (400 μm diameter) micro-molds were created by using custom casts fabricated by 3D printing (UV-resin; Formlabs 3D printer) as follows: 2% agarose and 1% sucrose in water was microwaved to completely dissolve; the mixture was poured into the 3D-printed cast and solidified at room temperature; and the resulting agarose micro-molds were equilibrated in culture media for at least 24 h. Approximately 10⁵ cells were seeded in the agarose micro mold. For the MoNNet samples, we generated custom polydimethylsiloxane(PDMS) molds, containing four 28 mm diameter wells, as follows: (1) custom cast were 3D-printed with acrylonitrile butadiene styrene (ABS; Ultimaker 2+), (2) fresh PDMS was prepared my mixing uniformly the silicone elastomer base and the curing agent at a ratio of 10:1 (weight/weight), followed by degassing in a vacuum chamber, (3) the freshly prepared PDMS was poured on to the cast and covered with a coverslip, (4) PDMS was cured at 90 °C overnight, (5) the resulting PDMS molds were sterilized by immersing in 100% alcohol overnight. Approximately 2 × 10⁵ cells were seeded in each of the four wells in the PDMS mold. For both individual isolated spheroids and MoNNets cultures, the cells were allowed to settle for 15 min, followed by addition of 2 ml of culture media. All cell cultures were kept in an incubator at 37 °C and 5% CO₂. Immunostainings with Caspase-3 were used to assess for any apoptosis in the preparations.