On the basis of the existing methods of primary culture of neuronal cells, we optimized the extraction steps and extraction practices, and used a reasonable gradient mechanical discrete method to obtain in vitro neuronal cell models with high purity and good activity. In this study, Sprague-Dawley (SD) rats within 24 h of birth were used as experimental subjects. Brain tissues were obtained by the guillotine method and the cerebral cortex was isolated. The tissue digestion process was performed using a two-enzyme combined digestion system of papain and deoxyribonuclease I (DNase I), which was terminated by adding 1 mL of fetal bovine serum after digestion at 37°C for 20-30 min. Subsequently, single-cell suspensions were prepared by gradient mechanical disaggregation, and the cells were finally inoculated into polylysine-coated six-well plates for subsequent experiments. At 4 h, 24 h, 48 h and 7 d after inoculation, the cells were observed under the microscope and photographed. On the 7th day, the neuronal cells were identified by immunofluorescence. The cortical neuronal cells extracted by this method showed a typical morphology, with a full cytosol, clear boundaries, abundant dendritic and axonal branches, and formed a complex neural network; the purity of the neurons identified by MAP2 immunofluorescence technique reached more than 95%. Compared with the traditional primary cortical neuron extraction method, this method utilizes multiple details such as dual-enzyme co-digestion system and gradient mechanical discretization method to explore and improve, and successfully establishes a stable result, simple and feasible primary cortical neuron culture method, which is a more ideal model for the study of neuroscience field.

Haiping Xu, Honglian Ya, Lanqing Meng. A rational distribution pipetting method for the separation and culture of primary cortical. Frontiers in Medical Science Research(2025), Vol. 7, Issue 2: 91-96. https://doi.org/10.25236/FMSR.2025.070213.

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