Primary Cortical Neuron Isolation from Rat Embryos

We purchased pregnant Sprague-Dawley rats (embryonic day 18; SAMTAKO) and euthanized perinatal embryos for isolating primary cortical neurons, following a previous protocol (29 (link)). We conducted all procedures according to the animal welfare guidelines approved by the Institutional Animal Care and Use Committee of the Korea Institute of Science and Technology. Briefly, we dissected the entire cortex as an intact structure from the embryos’ brains under a dissection microscope. Next, we treated the cortex with papain (Miltenyi Biotec) and serially triturated. Then, we seeded dissociated cells, predominantly neurons, within collagen; see the following section for more details. We cultured the neurons in medium consisting of neurobasal medium supplemented with 2% (v/v) B27 supplement (Invitrogen), 2 mM GlutaMAX I (Thermo Fisher Scientific), and 1% (v/v) penicillin-streptomycin (Thermo Fisher Scientific) in a humidified incubator at 37°C and 5% CO₂. During the 3D culture, we replaced half the medium with fresh medium every 2 to 3 days.