OBJECTIVE: To improve previous method of primary rat cortical neuron culture to get purer and more long-lasting cells for study. METHODS: Timed-pregnant Wistar rats at a gestational age of 16 or 17 days (16-17 d) were used. Fetal brains were removed and the cerebral cortices were dissected out. Papain digestion and mechanical dissociation were combined to conduct mono-cell suspending media. Four to six hours (4-6 h) post-plating, all plating media were removed from cultures and replaced with Neurobasal medium supplemented with B27. On the third day, 10 mumol/L cytosine arabinoside (Ara-C) was added to the culture for 24 h to inhibit the outgrowth of glial cells. Half of the culture medium was changed every week. The morphological changes of neuron cells were observed by light microscope. Double immuno-staining of microtubule-associated protein 2 (MAP2) and karyon were applied to assess the culture purity. Evaluation of synapse formation was processed by immunocytochemical analysis using antibodies against both pre- and postsynaptic protein markers. RESULTS: The improved method could remarkably increase the cell number and reduce neuronal damnification. The primary culture was characterized by high uniformity, purity, normal synapse formation and longtime livability. CONCLUSION: This is a simple and reliable technique for the in vitro primary culture of rat cortical neurons.
OBJECTIVE: To improve previous method of primary rat cortical neuron culture to get purer and more long-lasting cells for study. METHODS: Timed-pregnant Wistar rats at a gestational age of 16 or 17 days (16-17 d) were used. Fetal brains were removed and the cerebral cortices were dissected out. Papain digestion and mechanical dissociation were combined to conduct mono-cell suspending media. Four to six hours (4-6 h) post-plating, all plating media were removed from cultures and replaced with Neurobasal medium supplemented with B27. On the third day, 10 mumol/L cytosine arabinoside (Ara-C) was added to the culture for 24 h to inhibit the outgrowth of glial cells. Half of the culture medium was changed every week. The morphological changes of neuron cells were observed by light microscope. Double immuno-staining of microtubule-associated protein 2 (MAP2) and karyon were applied to assess the culture purity. Evaluation of synapse formation was processed by immunocytochemical analysis using antibodies against both pre- and postsynaptic protein markers. RESULTS: The improved method could remarkably increase the cell number and reduce neuronal damnification. The primary culture was characterized by high uniformity, purity, normal synapse formation and longtime livability. CONCLUSION: This is a simple and reliable technique for the in vitro primary culture of rat cortical neurons.