悬浮法培养C6胶质瘤细胞系和该细胞系中脑肿瘤干细胞的分离

C6 rat glioma cell line contains brain tumor stem cells and maintained in floating-culture system

目的研究应用无血清培养基悬浮法培养C6胶质瘤细胞系的方法;并分离该细胞系中的脑肿瘤干细胞,观察其生长方式和分化特征。方法应用培养大鼠神经干细胞的培养基和培养方法,在无血清培养基中采用悬浮法培养大鼠C6胶质瘤细胞系;再将分离获得的悬浮生长的脑肿瘤干细胞接种于含血清培养基,观察其分化;应用有限稀释和单克隆形成实验确定该细胞系中肿瘤干细胞的比例。结果C6胶质瘤细胞系中有约1.18%的肿瘤细胞在无血清培养基中能够存活、增殖,形成自由漂浮的细胞球;这种细胞球具有人脑肿瘤干细胞球的特征,并可连续传代,若重新接种于含血清培养基中可重新贴壁分化,贴壁分化后细胞形态与直接在含血清培养基中培养的C6胶质瘤细胞系无明显差别。结论C6大鼠胶质瘤细胞系中含有比例较低的、具有增殖和分化能力脑肿瘤干细胞(约1.18%),该细胞系可在含生长因子的无血清培养基中悬浮生长,并维持细胞系。

Objectives: To study whether the C6 rat glioma cell line contains brain tumor stem-like cells and its growing pattern in serum-free medium supplemented with mitogens. Methods: Adherent C6 cells in serum-supplemented medium were dissociated to single cell suspensions and were seeded in serum-free medium supplemented with FGF and bFGF in 6-well plates. After the C6 brain tumor spheres formed, they were dissociated and passaged periodically. Limiting dilution analysis and monoclonal formation assay were also performed. C6 brain tumor spheres or the single cells of them were washed and induced to differentiate in the medium with 10% FBS supplemented and EGF, bFGF withdrawal. Results: About 1.18%±0.10% (SD) of C6 glioma cells are clonogenic in C6 glioma cell line. They can generate brain tumor spheres in serum-free medium and the cell line can be maintained by passaging the brain tumor spheres for prolonged times. C6 glioma spheres and the single cells of them can be induced to differentiate and adherent to the bottom of the culture plates. The growing patterns of C6 glioma cell line can be converted by changing the culture mediums. Conclusion: C6 glioma cell line contains C6 glioma stem cells strongly resembling brain tumor stem cells that are isolated from human neuroepithelial tumors, and the cell line can be maintained in serum-free medium with floating-culture method for prolonged times.