The animal experiments and procedures were approved by the Institutional Animal Care Committee at Technion—Israel Institute of Technology and were in accord with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
We removed and diced cortical tissue from embryonic day 18 (E18) Sprague Dawley rats in cold PBS solution with 20 mM glucose, mechanically dissociated neural cells (by forcing a few times through a pipette), and filtered the suspension using a 70μm cell strainer (Biologix). 1.2-1.8 million cells were encapsulated in 30 μl Matrigel scaffold (Growth factor reduced, BD Biosciences). Culturing media was composed of Minimal Essential Media (MEM, Sigma) containing 17 mM glucose, 100μl/ml of NU Serum (BD Biosciences), 30mg/ml of L-Glutamine (Sigma), 1:500 B-27 supplement (Gibco), 50ng/ml of Nerve Growth Factor (NGF, Alomone labs), 10ng/ml of Brain-Derived Neurotrophic Factor (BDNF, R & D systems), 25 μg/ml of Insulin (Sigma), and 2μg/ml of Gentamicin. Half the volume of the culturing media was replaced twice a week. In a second group of cultures, 3μM of Arabinofuranosyl Cytidine (ARA-C, Sigma) to inhibit glial cell proliferation was added once at DIV 3.
We removed and diced cortical tissue from embryonic day 18 (E18) Sprague Dawley rats in cold PBS solution with 20 mM glucose, mechanically dissociated neural cells (by forcing a few times through a pipette), and filtered the suspension using a 70μm cell strainer (Biologix). 1.2-1.8 million cells were encapsulated in 30 μl Matrigel scaffold (Growth factor reduced, BD Biosciences). Culturing media was composed of Minimal Essential Media (MEM, Sigma) containing 17 mM glucose, 100μl/ml of NU Serum (BD Biosciences), 30mg/ml of L-Glutamine (Sigma), 1:500 B-27 supplement (Gibco), 50ng/ml of Nerve Growth Factor (NGF, Alomone labs), 10ng/ml of Brain-Derived Neurotrophic Factor (BDNF, R & D systems), 25 μg/ml of Insulin (Sigma), and 2μg/ml of Gentamicin. Half the volume of the culturing media was replaced twice a week. In a second group of cultures, 3μM of Arabinofuranosyl Cytidine (ARA-C, Sigma) to inhibit glial cell proliferation was added once at DIV 3.