were discarded. The single cell suspension of day 17–19 neurons was then frozen or directly plated at 62.5k cells/ 3.8 cm2 (one well of 12WP) in direct contact or indirect contact mode in the presence of astrocytes for maturation. To stop division of hNES cells and to facilitate neuronal maturation, the direct contact co-cultures were treated with 2 μM cytosine β-D-arabinofuranoside (AraC) at day 25 and 29. Neurons were refreshed twice a week with half of the Neurobasal medium containing BDNF, GDNF, IGF1 and cAMP. The concentration of the growth factors in the refreshment medium was doubled (enough for the whole well).
