Mouse glial cells, obtained from postnatal day 0 (P0) pups, were seeded into the devices on Day 5 (one day prior to neuron seeding). In the central chamber, 20,000 glial cells were seeded (∼300 glia/mm2). In the side chambers, 12,000 glial cells were seeded (∼210 glia/mm2). For neurons, 150,000 neurons were seeded in the central chamber (∼1,580 neurons/mm2) and 75,000 neurons were seeded in each side chamber (∼1,875 neurons/mm2). iNs were removed from the well plate surface using Accutase (Innovative Cell Technologies, San Diego, CA) and plated with Neurobasal media containing L-glutamine, B27, NT3 (10 ng/ml), GDNF (10 ng/ml), BDNF (10 ng/ml), 5% FBS, 1% penicillin/streptomycin and 2 μg/ml doxycycline. After cell attachment, and as needed during the course of the culture, 2 μM cytosine arabinoside (AraC) was added to the media to control glial cell division. Devices were fed every 2–3 days. Devices were housed individually in 35 mm culture dishes. Two 35 mm dishes were placed in a 10 cm culture dish and approximately 1 ml of sterile water or PBS was added to the 10 cm culture dish to limit evaporation of media.
