神经干细胞团的悬浮不稳定性及其解决方法  

Suspending Instability of Neural Stem Cell Clusters and its Solution

作　　者：郑学胜[1] 杨小锋[1] 刘伟国[1] 冯军峰[1] 胡未伟[1] 李谷[1] 罗鸣[1] 陈向日[1] 

机构地区：[1]浙江大学医学院附属第二医院神经外科,杭州310009

出　　处：《中国生物医学工程学报》2006年第5期607-612,共6页Chinese Journal of Biomedical Engineering

基　　金：浙江省科技厅重点资助项目(021106133);浙江省自然科学基金项目(Y205180)

摘　　要：目的探讨体外培养的神经干细胞团不能稳定悬浮的相关因素,建立一种适合神经干细胞长期、稳定悬浮培养的方法。方法在培养过程中动态检测培养基密度和粘滞系数,神经干细胞团密度和半径,同时观测神经干细胞贴壁现象。探讨调节培养基密度和粘滞性、利用琼脂糖凝胶包被培养瓶等方法的抗贴壁效果,及其对干细胞增殖和分化的影响。结果神经干细胞团半径逐渐增大,同时细胞团的沉降速率明显增大。传代第3天细胞团开始贴壁。而培养基密度、粘滞系数和细胞团密度均无明显变化。通过调节培养基密度和粘滞系数,使细胞团保持悬浮,但S期标记指数(LI)下降,细胞逐渐死亡。通过在培养瓶底包被凝胶隔离层,神经干细胞团保持快速增殖而不贴壁,长期培养分化率低。结论神经干细胞团的悬浮不稳定性主要与细胞团体积相关。通过琼脂糖凝胶包被培养瓶底,可以防止贴壁,有利于神经干细胞持续增殖并保持未分化,适合神经干细胞长期培养。Objective To investigate the reason that neural stem cell clusters cannot keep suspending in the culture medium, and develop a novel anti-attachment culture system for long-term neural stem cell cultivation. Methods The density,viscosity coefficient of the medium and the density, radius of cell clusters were measured during the 2nd～7th days after passage. The neural stem cells were studied morphologically everyday. In order to prevent cell clusters from attaching, two methods were used, including adjusting the medium＇s density arid viscosity and coating the flask bottom with agarose gel. The influence of these methods on the proliferation and differentiation of neural stem cells was studied using BrdU in corporation assay and immunocytochemistry. Results The radius of cell clusters increased gradually, and the settling velocity of the clusters in the medium increased accordingly. Neural stem cells began to attach 3rd day after passage. However, the density and viscosity coefficient of the medium and the density of clusters did not change significantly. When the medium was adjusted to increase its density and viscosity, cell clusters kept floating, however, S-phase labeling index（ LI） decreased and cells died gradually. When the flask bottom was coated with agarose gel, neural stem cells grew rapidly without attachment. After 3 months in culture, only 0.64% cells had differentiated, while most cells still maintained their muhipotency. Conclusions The suspending instability of neural stem cell clusters is mainly related to their volumes. Agarose gel coating method is suitable for long-term cultivation of neural stem cells, since it is in favor of cell proliferation and capable of preventing neural stem cell from attaching and differentiating.

关 键 词：神经干细胞 悬浮 贴壁 

分 类 号：R329.12[医药卫生—人体解剖和组织胚胎学]