OBJECTIVETo improve the efficiency of primary culture of hippocampal neurons and obtain highly purified neurons with good in vitro growth and minimal risk of contamination.

METHODSThe hippocampal neurons of neonatal Wistar rats were isolated and the single cell suspension was prepared by mechanical trituration and sedimentation in stead of trypsin digestion and filteration. Twenty-four hours after the cell plating, the culture medium was removed and replaced by serum-free DMEM/F12 with B27 supplementation. Half of the culture medium was changed 2-3 times every week. The morphological changes of the neurons were observed under inverted phase-contrast microscope. Immunofluorescence staining for NSE was performed to identify the neurons, and the purity of neurons was calculated. The hippocampal neurons were stained with calcium-sensitive fluorescent dye to monitor the effect of KCl on neuronal excitability by a calcium imaging system.

RESULTS AND CONCLUSIONThis simplified method is time-saving and cost-effective for primary culture of hippocampal neurons with reduced risk of contamination, and the neurons obtained showed high uniformity, purity and long-term viability.