关键词: 皮质神经元, 星形胶质细胞, DNA酶, 免疫荧光, 原代培养

Abstract: BACKGROUND: At present, there are many methods to obtain primary cortical neurons and astrocytes. The traditional method is usually to obtain these two kinds of cells separately, but the experimental method is too cumbersome and wastes experimental materials. It is particularly important to find a simple, economical, and feasible culture method that simultaneously extracts both types of cells.  
OBJECTIVE: To observe the effect and precautions of simultaneously extracting neurons and astrocytes from the cerebral cortex of Sprague-Dawley suckling rats.
METHODS: The suckling rats within 24 hours of birth were selected and sterilized with 75% alcohol. After the suckling rats were in a coma, they were sacrificed by cutting their spines. The head was severed with scissors along the neck of the suckling rats and placed in a small beaker containing high-glucose DMEM. The skull was opened through the suture and the brain was removed and placed in a petri dish containing high glucose DMEM. The meninges and blood vessels were peeled off on ice, and the brain tissue 2.0-3.0 mm on the surface of the cortex was clipped, digested with papain and DNase, and terminated with DMED/F12 containing 10% fetal bovine serum. Samples were seeded at a density of 1.0×109/L in six-well plates containing slides (treated with polylysine). The morphological changes of neurons were observed under an inverted fluorescence microscope at 1, 3, 5, and 7 days, respectively. Calcien-AM was used for live cell staining to observe cell viability, and β-Tubulin and MAP2 immunofluorescence staining was used to identify neurons. Astrocytes were extracted from the remaining cerebral cortex. After trypsin digestion, filtration, and pipetting, they were inoculated into culture flasks at a suitable concentration. When the cell confluency reached 90%, the astrocytes were shaken on a constant temperature horizontal shaker for 24 hours to purify the astrocytes. After the cells were passaged and purified, they were seeded in six-well plates at 1.0×109/L. Astrocytes were identified by GFAP immunofluorescence staining. 
RESULTS AND CONCLUSION: (1) After 1 day of culture, neurons could be seen to adhere to the wall; the cell body increased, and some of the cells grew together, and there were a few synaptic connections between cells. After 3 and 5 days, the cell body further increased, and the synapses were thickened and elongated to form a sparse network. After 7 days, the neurons were obviously aggregated; the synapses grew and thickened, and they connected to each other to form a dense cell network. Calcien-AM live cell staining showed better neuronal viability. The purity of the two identification methods was greater than 90% identified by β-Tubulin and MAP2 immunofluorescence staining. (2) After 3 days of culture, the astrocyte bodies were enlarged, showing spindle and irregular shapes, with a small amount of interconnected cells and many microglia between cells. After 5 days of culture, the cell bodies and protrusions further increased. After 7 days of culture, it was found that the microglia and other impurities between the cells were reduced compared with the previous ones, and the background was relatively clean. The purity of the cells was greater than 95% identified by GFAP immunofluorescence staining. (3) The obtained cortical neurons and astrocytes have good morphology, less impurities and high purity. They can meet the needs of high-purity cortical neurons and astrocytes for neuroscience research.

Key words: cortical neuron, astrocyte, DNase, immunofluorescence, primary culture