BrainPhys™ with L-glutamine and NeuroCult™ SM1 supplements with 1% P/S, 10 ng ml−1 BDNF, 10 ng ml−1 GDNF, 10 ng ml−1 NT3, and 2 μg ml−1 doxycycline. At day 8, 80 μl of media were removed from each well, followed by the addition of 100 μl of BrainPhys™ with supplements. On day 12, 100 μl were removed from each well and replaced with 100 μl of BrainPhys™ media with supplements. After day 12,100 μl were removed from each well and replaced with approximately 250 μl of fresh media. Some wells required the addition of extra media during each feeding due to their plate location resulting in greater media evaporation. Devices were cultured for 5–7 weeks prior to analysis. Mature cultures expressing GCaMP6f projected axonal bundles through the microchannels (Fig. 3).
