Improvement of primary culture method of rat spinal cord neurons

English Keywords:Spinal cord neuron  Primary culture  Enzyme digestion  Accutase enzyme method

English Abstract:

  【Abstract】 Objectives: To introduce a method to improve the culture technique of embryonic spinal cord neurons. Methods: 36 male and 36 female SD rats of childbearing age were mated and the female rats were pregnant. Firstly, six SD pregnant rats were selected respectively from each of the three groups of gestational ages of 10-12d, 13-15d, and 16-18d, and embryonic spinal cord were dissected and separated. Single cell suspension was prepared by routine trypsin digestion method. The total number of cells and the number of blue cells were counted under the microscope by cell counting plate and trypan blue staining, and the cell survival rate was calculated to determine the optimal gestational age of the material. Then, taking the above optimal gestational age as the sampling time point, six pregnant rats in each group prepared single cell suspension by three different digestion methods: enzyme free digestion, trypsin digestion and Accutase digestion. Using cell counting plate and trypan blue staining to count the total number of cells and blue cells under the microscope and calculate the cell survival rate. The isolated cells were inoculated, purified and cultivated for 1, 3, and 7 days. The growth vitality of the spinal neurons was observed under an inverted phase contrast microscope. The purity of neurons was identified and determined by double staining with the neuron-specific marker β-tubulin Ⅲ and nuclear marker. Results: The total number of cells and the number of viable cells in the 13-15d group were higher than those in the 10-12d group, and the cell survival rate in the 13-15d group was higher than that in the 16-18d group(P<0.05). The total number of cells, viable cells and cell survival rate in the Accutase group were significantly higher than those in the enzyme free group and the trypsin group(P<0.05). After culturing 7 days, the light ring gradually appeared around the neuron body and the neurites gradually extended to form a dense network under inverted phase contrast microscope. Compared with the enzyme free group and the trypsin group, the network of spinal cord neurons separated by the accutase enzyme method was more uniform and denser. After β tubulin Ⅲ and hoechst33342 double fluorescence staining identification, the purity of spinal cord neurons isolated and cultured in the Accutase group, enzyme free group and trypsin group was (90.29±2.55)%, (83.51±6.20)% and (88.10±4.07)% respectively with no significant difference(P>0.05). Conclusions: The best time for primary culture of rat spinal cord neurons was 13-15 days after pregnancy. Compared with conventional enzyme free and trypsin free methods, Accutase enzyme method can obtain more spinal cord neurons with better growth status, providing a good cell model for the study of spinal cord neuropathy in vitro.