Abstract: Abstract: 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 Hippocampus of newborn rats(24 hours) were taken out and digested. Hippocampal neurons were planted in 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, a typical neuronalmorphology appeared on the third day. Up to the 5th day, many neurites extended to form dense network. Soma of neurons became were well developed on the 7th day. Fluorescence staining with β-tublinⅢ showed that the purity of neurons was 94.2±3.6%. Conclusion The hippocampal neurons with high purity can be obtained by the simple and efficient method.

Key words: Key words: hippocampus, neurons, primary culture, newborn rats