应用旋转生物反应器培养小鼠胚胎干细胞的初步研究

Proliferation and differentiation of mouse embryonic stem cells in rotary cell culture system

目的应用旋转生物反应器(RCCS)和微载体培养体系尝试建立一种实现批量培养干细胞的新方法。方法应用RCCS和微载体培养体系对小鼠胚胎干细胞(mESCs)进行体外培养扩增,定期收集细胞样品,镜下观察mESCs在RCCS生长的形态特征,并定量绘制细胞生长曲线,利用MATLAB软件计算细胞生长参数并对照平面培养体系,利用H&E染色、免疫荧光及RT-PCR技术对RCCS内培养的mESCs的细胞形态,未分化标志蛋白(SSEA-1)和标志基因(oct-4)的表达进行定性或半定量分析。结果mESCs可在RCCS内以贴附于微载体表面的形式实现三维生长,其生长增殖状态良好,且伴随培养时间的延长,SSEA-1蛋白及oct-4基因的表达水平逐渐降低。这表明RCCS内培养扩增的mESCs逐渐走向分化,该分化进程同步于平面对照培养体系。结论RCCS可以为mESCs的体外规模化扩增培养提供良好的培养体系。

Objective To establish a novel culture approach of scalable expanding stem cells in vitro by using rotary cell culture system （RCCS） and microcarriers. Method mESCs and microcarriers were seeded into RCCS and samples were collected at indicated time. The growth profile of mESC in RCCS was observed under phase contrast microscope. Their growth curve was obtained by Typan blue exclude staining. To evaluate the effect of RCCS on mESC phenotype, the expression of marker protein SSEA-1 and marker gene oct-4 were evaluated by immtmo-fluorescence staining and RT-PCR, respectively. Result ESC grew well in RCCS. As time passed the expression level of SSEA-1 protein and oct-4 gene decreased significantly, which was similar to the control culture system in petri-dish. This demonstrated that no negative effect was generated on mESCs capability in spontaneous differentiation. Conclusion RCCS could serve as an ideal three dimensional culture system for mESC proliferation and differentiation in vitro.