原代神经元与星形胶质细胞的混合培养方法

A protocol for primary dissociated astrocyte and neuron co-culture

原代培养的海马神经元是研究神经细胞中蛋白运输与亚细胞空间定位的有效研究工具。本文旨在建立海马神经元与皮层胶质细胞混合培养的方法,以期提供状态良好的神经元供研究用。新生2～3 d Sprague-Dawley(SD)大鼠断头处死,从大脑半球表面片取2～3片皮层组织,切碎后用胰蛋白酶消化成单细胞悬液,种植于25cm2培养瓶内。种植后第四天,采用剧烈敲击培养瓶的方法去除非星形胶质细胞的杂质细胞,继续培养贴壁细胞直至细胞接近铺满瓶底,改用含阿糖胞苷的神经细胞培养液。原代培养的大鼠海马神经元种植在这种预先培养好的皮层星形胶质细胞层上。镜下观察结果显示,本方法培养的原代星形胶质细胞纯度较高,共培养的神经元状态较佳,存活时间长,突触前结构发育良好,可以耐受转染操作。以上结果提示,本文所建立的原代神经元与星形胶质细胞的混合培养方法操作简单,能够可靠地培养出健康的神经元。

Cultured primary hippocampal neurons are ideal tool for investigating the subcellular localization and trafficking of neu- ronal proteins. The aim of the present study was to establish a method to co-culture hippocampal neurons and cortical astrocytes, which would guarantee well conditions of neurons. Newborn Sprague-Dawley （SD） rats were sacrificed by decapitation. The cortex of cerebrum was cut into pieces, and the cortical tissue was digested with trypsin. The liquid suspension of single cells was planted onto a 25 cm2 culture flask. On the fourth day of culture, the tissue cells except astrocytes were removed by intensive agitation of culture flask. Purified astrocytes were allowed to grow continuously until they reached most area of flask. At this time point, we replaced the culture media with neuronal cell media containing cytarabine, and planted the primary culture of rat hippocampal neurons onto the feed layer of cortical astrocytes. The microscopic observation results showed that, the astrocytes evenly grew without obvious bound- aries between each other, and exhibited good purity. The co-cultured hippocampal neurons were in good condition, developed inter- twined network of axons and dendrites, lived for a long time, and could tolerate gene transfection. Above all, this method is relatively simple from a technical point of view, yet provides healthy and reliable neuronal culture.