Optimal acting time of cytarabine in primary culture of rat cortical neurons / 中国组织工程研究

ABSTRACT

BACKGROUND:Toxic cytarabine is often used to prepare highly purified neurons in experimental studies addressing central nervous system diseases. However, the intervention time of cytarabine is little reported. OBJECTIVE:To determine the optimal intervention time of cytarabine(final concentration 10μmol/L) in primary culture of rat cortical neurons. METHODS:Rat primary cortical neurons were cultured in Neurobasal+B27 medium, and 10μmol/L cytarabine were added at 12, 24, 36 and 48 hours after culture, respectively. Half of the medium were changed every 48 hours. The morphology of neurons was observed under inverted microscope at 7 days. The purity and differentiation of neurons maturity were identified by immunocytochemistry method of neuron specific enolization enzyme staining. Morphometric analysis for all neuron-specific enolase positive cells was performed by Multifunction Computer Image Analysis System. RESULTS AND CONCLUSION:After addition of cytarabine at 24 hours of culture, the purity of neurons was more than 90%, well-differentiated cortical neurons accounted for 89.00%, and the area of neuronal body was the largest with the longest synapses. There were more neuron cells with transparent cytoplasm, and large nucleus. The cell body had good refraction and strong stereo sense. The neurons with 3-4 synapses and 3-4 bifurcates formed a good network structure. These results illustrate that although it willbe beneficial for the purity of neurons to add cytarabine early in neuron culture process, it will make obvious effect on neuronal differentiation. The highly purified and well-grown cerebral cortical neurons will be obtained after cultured in neurolbasal medium, which cytarabine is added to at 24 hours of culture.