Human neural progenitor cell culture

The v-myc immortalized human neural progenitor cell line ReNcell VM (EMD Millipore) was used in this study at between passages 4 to 8. Undifferentiated hNPCs were maintained by culturing on laminin (Sigma) coated T25 or T75 flasks in complete growth medium, consisting of ReNcell Maintenance Medium (EMD Millipore) containing 20 ng/mL FGF2 (EMD Millipore), 20 ng/mL EGF (EMD Millipore), and 100 U/ml penicillin-streptomycin (Gibco). Cells were passaged using Accutase and re-plated at 10,000 cells/cm² when approximately 90% confluent. Medium was changed the day after passaging and every second day after that.

For off-chip 3D culture, freshly passaged undifferentiated ReNcell VM were mixed with Matrigel (High Concentration Growth Factor Reduced, Corning) at such a ratio that the final cell concentration was 5 × 10⁶ cells/mL and the final Matrigel concentration was 1% (w/v). 100 μL of this cell-Matrigel suspension (containing 500,000 cells) was seeded into 8-well chamber microscope slides (Nunc) and incubated at 37 ^oC for 15 min to gel. Following, 400 μL media was added and exchanged every second day. All cultures were maintained within incubators at 37 ^oC and 5% CO2.