Ma Yue, Li Wen, Li Zhenshu, Lü Xin, Wang Xinyan, Huang Guowei
Department of Nutrition and Food Science, School of Public Health, Tianjin Medical University, Tianjin 300070, China.
Wei Sheng Yan Jiu. 2019 Sep;48(5):795-798.
To establish an effective method of primary and passage cultured cerebral cortical astrocyte of SD neonatal rat in vitro.
Cerebral cortex of two 2-day-old SD rats were taken with aseptic operation and then were cut to pieces. After stripped the pia mater, digested by 500 μL 0. 25% trypsin at 37 ℃ for 15 min. Next, dispersed cell suspension was made by mechanical method and filtered. Cell suspensions were incubated in an uncoated culture bottle at 37 ℃ for 15 min. The cells were inoculated at 5×10~6/m L in the T75 culture flask coated with L-polylysine. The cells were shaken at 200 r/min 37 ℃ for 18 h, then added 1 m L of trypsin to digest cells and then collected the cells. The morphology of the passage cells was observed under inverted phase contrast microscope, and the purity of astrocytes was identified by immunofluorescence staining of GFAP. The proliferate activity of passage cells was determined by MTS assay.
The purity of astrocytes was( 97. 86 ± 0. 91) %, and the growth and proliferation activity of astrocytes were good after passage.
A rapid, economical and effective method for obtaining astrocytes in the cerebral cortex of newborn rats was established.
建立一种体外原代及传代培养SD新生大鼠大脑皮质星形胶质细胞的有效方法。
无菌操作取2只2日龄SD大鼠的大脑皮质，剪碎，去除软脑膜后，于37℃用500μL 0.25%胰蛋白酶消化15分钟。接着，通过机械方法制成细胞悬液并过滤。将细胞悬液置于未包被的培养瓶中于37℃孵育15分钟。将细胞以5×10⁶/mL接种于用L-聚赖氨酸包被的T75培养瓶中。细胞于37℃、200转/分钟振荡培养18小时，然后加入1mL胰蛋白酶消化细胞并收集细胞。在倒置相差显微镜下观察传代细胞的形态，通过GFAP免疫荧光染色鉴定星形胶质细胞的纯度。用MTS法测定传代细胞的增殖活性。
星形胶质细胞纯度为(97.86±0.91)%，传代后星形胶质细胞生长及增殖活性良好。
建立了一种快速、经济且有效的获取新生大鼠大脑皮质星形胶质细胞的方法。
