一种小鼠背根神经节原代神经元细胞分离与培养的优化方法

An optimized method for isolation and culture of primary neurons from mouse dorsal root ganglion

目的 探索一种可以大量快速培养活力好且卫星胶质细胞污染少的原代神经元细胞的方法。方法 混合酶溶液消化体式显微镜下提取的背根神经节(DRG),随后接种于纤维连接蛋白提前包被的培养皿中,8 h后更换含5-氟尿嘧啶的维持培养基,抑制非神经元细胞增殖。结果 分离培养的感觉神经元细胞在培养过程中可维持15 d左右,部分细胞可维持更长时间。原代细胞于第3天即可观察到交错的轴突网络,接种细胞密度大,卫星胶质细胞污染情况少,细胞在7 d之内可投入使用,操作者经短时间训练后即可快速掌握方法。结论 新方法在传统方法的基础上进行了改良,可获取较多数量细胞活性好、纯度高的原代神经元细胞,相较传统方法具有可操作性高,重复率高的优势。

Objective To explore a method that can rapidly cultivate a large number of primary neurons with good vitality and less pollution of satellite glial cells.Methods Dorsal root ganglion(DRG)extracted under microscope was digested with mixed enzyme solution,and then inoculated into fibronectin coated culture dish.After 8 hours,the maintenance medium containing 5-fluorouracil was changed to inhibit the proliferation of non-neuronal cells.Results The isolated and cultured sensory neuron cells could last for about 15 days,and some of them could last longer.On the third day,the primary cells could observe the crisscross axon network.The density of the inoculated cells was high,and the pollution of satellite glial cells was less.The cells could be put into use within 7 days.The operator could quickly master the method after a short period of training.Conclusions The new method is improved on the basis of the traditional method,which can obtain a large number of primary neurons with good cell activity and high purity.Compared with the traditional method,the new method has the advantages of high operability and high repetition rate.