Establishment of a simple and convenient method for primary culture of cortical neurons and identification of culture results

TANG Shi-jun; ZHAO Dong; ZHU Li-cang; LI Xiao-tian; ZHU Wen-xue; YANG Peng; WANG Ye-zhong

doi:10.3969/j.issn.1002-266X.2015.48.006

Abstract：Objective To establish a simple and convenient method for primary culture and to identify its validity . Methods The cortical tissues of SD rats born within 24 h were collected , and we got the neuronal cell suspension by tryp-sin digestion , explanted onto poly-L-lysine-coated plates with DMEM +HG +10%FBS culture medium for 4 h, then the medium was replaced with neuronal culture medium with Neurobasal 2% B27+0.5 mmol/L glutamine.We observed the neuronal morphological changes for 8 days through inverted phase contrast microscope .Under the laser scanning confocal microscopy , neurons were cultured for 7 to 8 days, and the cells were identified by immunofluorescence staining .The den-sity and purity of neurons were calculated .Results When the cortex neurons were inoculated , they were small, bright, translucent, round, and scattered in the distribution .After inoculation, a small amount of cells adhered to the wall , 2 hours later, more cells adhered to the wall, and a few cells produced short neurites;4 hours later, most cells adhered to the wall, and a large number of cells produced neurites around the halo .After 3 days of cluture , the neurites were elonga-ted, intertwined, transparent, and had strong three-dimensional sense with the round , oval and fusiform shapes .After 5 to 6 days, the cell bodies of the neurons increased , and the processes of the neurons wove into a network .After 7 to 8 days, the body of neuronal cell was full , cytoplasm was translucent , nucleus was large and obvious , the cell body had strong re-fraction, three-dimensional sense was good , and the processes wove into a network structure .The neuron density was 1.5 × 106/mL and the purity was more than 90%.Conclusion A simple and convenient method for primary culture was estab-lished with high purity and high density , which can provide a good cell model for the related scientific research .