大鼠海马神经元及星形胶质细胞的体外培养

目的：改进大鼠海马神经元、星形胶质细胞及两者混合状态的体外培养方法，获得高纯度的海马神经细胞。方法：选取孕期18~20 d 的SD 大鼠胎鼠作为原代海马细胞培养的材料来源。断头后于冰板上剥离双侧海马，去除表面血管及薄膜，利用眼科剪分离为小块。随后使用冷平衡盐溶液洗涤3 次，将海马组织碎块移入酶消化液中。本方法选择Accutase 和0.1% DNase 作为酶消化液，37 ℃消化 15 min，期间轻柔振动。使用冷平衡盐溶液洗涤3 次，终止消化。0.1% DNase 加入Neurobasal 和B-27 的混合液对消化后的海马组织碎块进行轻柔吹打，获得单细胞悬液。将悬液过200 目筛，进行细胞计数。将过筛的单细胞悬液移入DMEM 培养基（DMEM+10%胎牛血清）中，按照每平方厘米2.5×104 的细胞密度进行接种。经PDL 孵育的塑料片置于培养基底部。细胞于37 ℃、5% CO2 培养箱内培养。4 h 后，依据所需细胞种类的不同，更换相应培养基。海马神经细胞于培养后7~10 d 可用于实验。结果：通过本方法可得到高纯度的大鼠海马神经细胞，且根据实验需求，通过更换培养基，可得到纯神经元、纯胶质细胞及两者混合的三种培养状态。培养的神经元及星形胶质细胞纯度均>98%。结论：该文在传统海马神经细胞体外培养方法基础上，进一步降低了实验操作难度，方法稳定有效。

Culture Method of Rat Fetal Hippocampal Neurons and Astrocytes

Objective：To develop an improved culture method of neurons and astrocytes from hippocampus of SD rat fetuses in order to aquire high purity hippocampal cultures. Methods： The cultures of primary hippocampal neurons and astrocytes were prepared from SD rat fetuses at gestation day 18~20. After decapitation，the rat embryonic hippocampal tissue was taken out， sepatated and cutted into cubes quickly on ice. Then hippocampal cubes were transferred to the dissociation enzyme slolution after being washed in HBSS for three times. We chose Accutase and 0.1% Dnase as the digestion enzymes. The hippocampal tissue was incubated at 37 ℃ for 15 minwith gentle shaking. Tissue digestion was stopped by being washed with HBSS for three times.
After digestion，the tissue was gently triturated with triturating solution containing Neurobasal， B-27 and 0.1% DNase. The cell suspension was passed through sterile nylon gauze （80 mm）. The hippocampal cell suspension was plated in 24-well plates coated with poly-D-lysine at a density of 2.5 × 104 cells·cm-2. The cultures were maintained at 37 ℃ in a humidified atmosphere of 5% CO2 in DMEM medium supplemented with 10% Fetal Bovine Serum. After 4 h of initial plating，the medium was replaced with different medium according to targeting culture status（ pure neurons，pure astrocytes or mixed culture）. After 7~10 days in culture，the rat hippocampal cells could be used in follow-up experiments. Results：With this method，we aquired high purity hippocampal culture from rats. By changing different medium，we aquired different culture status，pure culture of neurons or astrocytes，or mixed culture of both cell types. The purity of pure culture cells was higher than 98% （both neurons and astrocytes）. Conclusion：We developed an improved culture method based on traditional cultivation. This method has lower difficulty in operation and higher stability.