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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 obtain high purity spinal cord derived astrocytes. Methods Primary culture and subculture of mouse spinal cord glial cells were performed. Sterile conditions after 1 ~ 3dICR (Institute of Cancer Research) mouse spinal cord, after digestion trypsin graded digestion, combined with mechanical blow to the cells dispersed, made of single cell suspension, inoculation in the absence of substrate adhesion Glass culture flask, set at 37 ℃, 5% CO2 incubator. The culture medium is DMEM / F12 mixed culture solution. After culturing for 9-12 days, the cells are 80% -90% confluent and then shaker purification is performed. Discard the cell suspension containing exfoliated cells to remove oligodendrocytes and microglia and continue to culture until the cells are covered with the bottom of the bottle Passaged, and passaged cells were observed morphological and astrocyte immunofluorescence identification, GFAP immunocytochemical staining positive cells up to 98%. The results obtained by a constant temperature oscillation and passage of typical morphology, high levels of spinal cord-derived astrocytes. Conclusion High purity spinal cord derived astrocytes can be obtained with no substrate attachment, constant temperature oscillation and passage method.