Improvement of primary cortical neuron culture and establishment of oxygen-glucose deprivation model

Objective To establish a stable and efficient primary culture method for cortical neurons obtained from pregnant Sprague-Dawley rats and oxygen-glucose deprivation(OGD) model. Methods Cerebral cortex isolated from E17-18 day SD rats was dissociated to single cells by TrypLE express enzyme, and was proceeded to culture medium containing neurobasal supplement with B27. The morphology of cortical neurons was observed during growth process, and immunofluorescence was applied to identify the cortical neurons. After 7 days, cell viability was assessed by MTT assay while cytotoxicity determined by lactate dehydrogenase(LDH) immediately after OGD. Results Neurons grew well with distinct outline and bright halation. Immunofluorescence showed that the cultured cells were cortical neurons with high purity(93.2±1.3)%. Cortical neurons were exposed to OGD condition for 4, 5 and 6 h, and the cell viability was significantly reduced in a time-dependent manner at(52.7±1.3)%,(26.5±3.3)% and(20.8±1.1)%, respectively. However, LDH release level increased time-dependently at(52.9±4.8)%,(89.3±1.3)% and(93.2±1.1)%, respectively. Conclusion The method is simple, reliable and practical for culturing primary cortical neurons with high purity. The cortical neurons exposed 4 h to OGD is choosen as the best condition for OGD model using Gas Pack Pouch bag.