Serum-free primary culture and identification of hippocampal neurons from newborn rats

Objective To establish a simple and practical method of the serum-free primary culture of hippocampal neurons in vitro to obtain highly purified and energetic neurons.Methods Hippocampi of newborn rats after birth in 24 hours were taken out and digested.Hippocampal neurons were planted on the glass slides covered with Matrigel basement membrane.Twenty-four hours after the cell being plated,the culture medium was removed and replaced by serum-free neurobasal one with N2 and B27 supplementations.The morphological changes of the neurons were observed under inverted phase-contrast microscope at different time.Immunofluorescence staining for β-tublinⅢ was performed to identify the purity of neurons.Results A large number of hippocampal neurons began to adhere to the glass slides and develop small neurites in 3~24 hours.Then,cells with typical neuron morphology appeared on the third day.Up to the 5th day,many neurites extended to form dense network.Soma of neurons became well developed on the 7th day.Fluorescence staining with β-tublinⅢ showed that the purity of neurons was 94.2%±3.6%.Conclusions The present protocol is a simple and efficient method for culturing hippocampal neurons with high purity.