新生大鼠皮层神经元分离和培养方法的优化

Optimization of extraction and culture methods of cortical neurons in neonatal rats

目的:优化新生大鼠皮层神经元提取、培养的方法,以获得高纯度并且生长状态良好的神经元,为神经系统的体外研究提供实验技术。方法:取新生24 h内的SD大鼠皮层组织,通过单次消化法、分步消化法两种不同的消化方法制备单细胞悬液,并以3种不同接种密度铺板,采用Neurobasal-A培养基+2%B27+0.5 mmol/L L-谷氨酰胺的体系进行培养。通过台盼蓝染色计数的方法计数两种消化方法得到的单细胞悬液中总细胞数和活细胞数,并计算细胞成活率。采用分步消化法制备单细胞悬液,用倒置显微镜观察不同密度接种后的神经元培养至7 d的生长状态,并观察以适宜密度接种的神经元培养1~7 d的生长状态。分别用神经元核抗原(NeuN)和神经元特异性烯醇化酶(NSE)两种神经元标志物鉴定神经元纯度。结果:分步消化法得到的细胞数量及细胞成活率均明显高于单次消化法(P <0.01);以5×105细胞/孔的密度接种细胞得到的神经元生长状态良好,密度适宜,形成的突触网络丰富而均匀;神经元培养1~7 d,胞体逐渐增大,突触逐渐伸长,直到第7 d,神经元胞体立体饱满、折光性强,形成均匀密集的突触网络;经鉴定,培养的神经元纯度可达95%以上。结论:分步消化法提取神经元、接种密度为5×105细胞/孔的培养稳定、高效,得到的神经元纯度高、生长状态好,可作为神经系统疾病体外研究的良好细胞模型。

Objective: To optimize the methods of extraction and culture of cortical neurons in neonatal rats,to obtain neurons with high purity and in good state of growth,and to provide experimental techniques for the study of nervous system in vitro. Methods: The cortical tissue of SD rats within 24 hours was isolated,and the single-cell suspension was prepared by one-step digestion and multi-step digestion. Three different cell densities were used to inoculate neurons respectively. Neurobasal-A medium + 2% B27 + 0. 5 mmol/L L-glutamine was used for culture. The total number of neurons and living neurons in the single-cell suspension obtained by the two digestion methods were counted by trypan blue staining,and the cell survival rate was calculated. The growth state of neurons inoculated with different densities was observed by inverted biological microscopy at 7th day,which were prepared by multi-step digestion. The growth state of neurons inoculated with suitable density for 1 ~ 7 days was observed,too. The purity of neurons was identified by neuronal nudear antigen (NeuN) and neuron-specific enolase (NSE) neuron markers respectively. Results: The number and survival rate of cells obtained by multi-step digestion were significantly higher than those by one-step digestion. The neurons grew well with density of 5 × 105 cells/well,which was suitable,and the synaptic networks were rich and uniform. 1 ~ 7 d of neuron culture,neuronal bodies enlarged and synapse lengthened gradually. Until the 7th day,neuronal bodies was full and refractive,formed a uniform and dense synaptic network. The purity of cultured neurons was identified as more than 95%. Conclusion: The culture method of multi-step digestion and inoculation density of 5 × 105 cells/well is stable and efficient. The neurons obtained with high purity and in good growth state,which can be used as a good cell model for the study of nervous system diseases in vitro.