[An improved method for primary culture of rat cortical neuron and cell identification].

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.