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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. [ABSTRACT FROM AUTHOR]

BACKGROUND: In recent years, primary neuronal cell models have played an important role in brain diseases. The characteristics of such cell models are closer to disease cells and can be used to simulate various neurological diseases. OBJECTIVE: To establish a convenient and practical culture method that can simultaneously extract primary cortical and hippocampal neurons. METHODS: The SD rats within 24 hours of the newborn were sacrificed by chiropractic method, and then sterilized with 75% ethanol. After separating the skull and meninges using forceps, the whole brain was dissected out. The cerebrovascular membrane was stripped, and the cerebral cortex and hippocampus were dissociated. The sequential digestion protocol of papain and appropriate amount of DNase was used. After pipetting, centrifugation, and filtration, the samples were inoculated into L-polylysine-coated six-well plates and slides. DMEM-F12 medium containing 10% fetal bovine serum was used as the seeding medium. 6 hours later, the serum-free special medium containing Neurobasal B27 was used. After culturing for 7 days, the cell morphology and growth state were observed under the microscope. The cortical and hippocampal neurons were identified by β-Tubulin immunofluorescence method and Neun antibody immunohistochemistry. The marker MAP2 immunofluorescence method was applied to identify the purity. RESULTS AND CONCLUSION: (1) 24 hours after inoculation, the volume of cells became clear, presented irregular circles, surrounded by halos, and a few cells had small protrusions, all of which had grown adherently to the wall. After 3 days of continuous culture, the cell bodies gradually increased; some of them grew in clusters; synapses were elongated; and cross-links appeared between cells. After 7 days of continuous culture, the cell body was mature and full; the cytoplasm was significantly increased; the halo was enhanced; and a denser neuronal network was formed. (2) Neurons were identified by Neun antibody immunohistochemistry and β-Tubulin immunofluorescence method. The purities of cortical and hippocampal neurons were (94.00±0.34)% and (91.00±0.26)%, respectively detected using neuronal marker MAP2 immunofluorescence method. Neuronal cells could be used for experiments. (3) These results suggest that the cerebral cortex and hippocampus were isolated from the same batch of neonatal SD rats within 24 hours. After sequential digestion with papain and DNase, high-quality hippocampal and cortical neurons can be extracted. The neurons obtained by this protocol have high purity and simplified operation, and can be used as the basis for various neurological disease cell models. [ABSTRACT FROM AUTHOR]