A Modified Method for Primary Culture of Newborn Rat Cortical Neurons and Its Basic Electrophysiological Property

Objective:To modify previous method of primary newborn rat cortical neuron culture to get purer and more long-lasting cells for some related studies.Methods:The cerebral cortices were aseptically removed from 0-24 h-old SD rats and dissected out.Mechanical dissociation and trypsin digestion were combined to conduct mono-cell suspending media.The dissociated momo-cortical cells were diluted to 1×105/ml then to be plated on poly L-lysine-coated petri dish.After 24h culture,3μg/mL cytosine arabinoside(Ara-C) was added to the culture for 24 h to inhibit the outgrowth of glial cells.After three days post-planted,all media were removed from cultures and replaced with medium supplemented with B27.Then half of the culture medium was changed every three days.The morphological changes of neuron cells were observed by light microscope.Immuno-staining of microtubule-associated protein 2(MAP2) was applied to assess the culture purity.Single channel current was recorded to evaluate the neurons properties by using patch clamp technique.Results: The improved method could increase the cells lifetime.On the 36th day,the primary culture was characterized by normal morphology and was gained typical BKca current.Conclusion:This is a simple and reliable technique for the in vitro primary culture of rat cortical neurons which have longtime livability and normal electrophysiological property.