2023, Vol. 27 ›› Issue (6): 897-902

An experimental method for simultaneously culturing primary cortical and hippocampal neurons

Liao Yidong1, Ming Jiang1, Song Wenxue1, Wang Zili1, Zhang Yu2, Liao Yifei2, Xu Kaya2, Yang Hua2   

Abstract: 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.
Key words: neonatal SD rat, cortical neuron, hippocampal neuron, primary culture, neurological disease, cell model