小鼠脊髓原代星形胶质细胞培养方法的建立

Culture of primary astrocytes from Spinal Cord of mice

目的获得纯度较高的脊髓源性星形胶质细胞。方法采用小鼠脊髓神经胶质细胞的原代培养和传代培养。无菌条件下取生后1～3dICR(Institute of Cancer Research)小鼠的脊髓,剪碎后胰酶分次消化,结合机械吹打使细胞分散,制成单细胞悬液,接种于无底物黏附的玻璃培养瓶中,置37℃,5%CO2培养箱中培养。培养液为DMEM/F12混合培养液。培养9～12d待细胞达到80%～90%融合后进行摇床纯化,舍弃含脱落细胞的细胞悬液,以去除少突胶质和小胶质细胞,继续培养,待细胞铺满瓶底后传代,并对传代细胞进行形态观察和星形胶质细胞的免疫荧光鉴定,GFAP免疫细胞化学染色阳性细胞可高达98%。结果通过恒温振荡和传代后得到了形态典型,含量较高的脊髓源性星形胶质细胞。结论用无底物贴附,恒温振荡和传代方法可获得高纯度脊髓源性星形胶质细胞。

Objective To purify astrocytes from spinal cord for further experiments.Methods Primary culture and passage of astrocytes in vitro.Under sterile environment the spinal cord was obtained from ICR mice born in 1～3 days,dissected and prepared cell suspension by digestion with the use of trypsin and mechanical digestion,then transferred to culture flasks without any stratum,and cultured（37℃,5%CO2）.The culture medium was DMEM/F12 and then reduced oligodentrocytes and microglial cells with orbital shaker when it was confluent by 80%～90% on days 9 through 12.The primary culture was passaged when it was confluent.Finally the secondary culture was observed and the astrocytes were identified by immunohistochemical staining with anti-mouse GFAP.The GFAP-positive cells were counted no less than 98%.Results By orbital shaker in constant temperature and pasaage the purified astrocytes with typical shapes were got.Conclusion With orbital shaker in constant temperature,passage and cultivation without any stratum,we successfully obtained the high purity astrocytes from neonatal mice spinal cord.