小鼠大脑皮质星形胶质细胞的体外培养方法及优化

Optimization and methods of culture in vitro of astrocytes from cerebral cortical mice

目的探究并优化新生小鼠大脑皮质星形胶质细胞体外原代培养方法,为星形胶质细胞的体外培养提供更优解。方法为优化小鼠大脑皮质星形胶质细胞体外培养方法,取新生3 d的C57BL/6J小鼠大脑皮质组织,去除脑膜和血管,经胰酶消化后离心,加入高糖的杜氏改良Eagle培养基(DMEM)吹打形成细胞悬液。进一步通过差速贴壁法、十字交叉手摇法以及恒温振摇法进行纯化,将细胞分别以不同培养密度接种于多聚赖氨酸包被的培养瓶中,并通过形态学观察,免疫荧光染色等方法对星形胶质细胞进行纯度鉴定。结果新生小鼠大脑皮质细胞以5×10^(6)个/瓶密度接种效果好,活性高;纯化过程中使用高糖DMEM培养基联合差速贴壁法、十字交叉手摇法及恒温振摇法,星形胶质细胞纯度可达99%。结论成功建立并优化小鼠大脑皮质星形胶质细胞的原代培养方法。

Objective To explore and optimize the in vitro primary culture method of astrocytes in neonatal mouse cerebral cortex,which provides a better solution for the in vitro culture of astrocytes.Methods In order to optimize the in vitro culture method of mouse cerebral cortex astrocytes,3-day-old C57BL/6J mouse cerebral cortex tissues were taken,meninges and blood vessels were removed,digested by pancreatic enzymes and centrifuged,and high-glucose dulbecco′s modified eagle medium(DMEM)was added to form cell suspension,which was purified by differential adhesion method,cross hand method and constant temperature shaking method.The cells were inoculated in poly-D-lysine-coated culture bottles with different culture densities,and the purity of astrocytes was determined by morphological observation and immunofluorescence staining.Results The cells were inoculated at a density of 5×10^(6)cells per bottle with good effect and high activity.The purity of astrocytes reached 99%by using high sugar DMEM medium combined with differential adhesion method,cross hand method and constant temperature shaking method.Conclusion The primary culture method of astrocytes in mouse cerebral cortex is successfully established and optimized.