小鼠胚胎干细胞在六种培养体系的培养观察

A Study on Six Kinds of Cell Culture System for Mouse Embryonic Stem Cells

目的 观察小鼠胚胎干细胞在六种培养体系中的生长情况。方法 小鼠胚胎干细胞 (ESD3细胞株 )在以下六种培养体系中培养 :1 .原代小鼠胚胎成纤维细胞 (MEF)有血清培养 ,2 .MEF无血清培养 ,3.SNL细胞有血清培养 ,4.LIF(白血病抑制因子 )有血清无饲养层培养 ,5.LIF无血清无饲养层培养 ,6.大鼠肝细胞 (BRL)条件培养基培养。经体外培养 1 0代后 ,观察其克隆形态 ,同时进行碱性磷酸酶检测并将ES细胞接种于裸小鼠皮下 ,观察ESD3的未分化状态和多潜能性。结果 六种培养体系培养的ESD3具有典型的ES细胞克隆形态 :巢状 (集落状 )隆起生长 ,边缘清楚 ,表面平滑 ,结构致密 ;AKP强阳性 ;裸小鼠体内形成了由多种组织构成的畸胎瘤。结论 六种培养体系均能支持ESD3生长 ,并能保持其未分化性和多潜能性 ,为ES细胞的应用研究奠定了良好的基础。

Objective To establish six kinds of stable embryonic stem cell culture system. Methods Mouse embryonic stem cells (ES D3 line) are maintained in following culture systems: The primary mouse embryonal fibroblasts (MEF) in serum medium and MEF in serum-free medium as well as SNL (mouse fibroblast subline ) in serum medium all together consist three kinds of feeder layer dependent culture systems. The serum medium and serum-free medium supplemented with recombinant murine leukemia inhibitory factor (mLIF) respectively as well as Buffalo Rat Liver (BRL) conditioned medium consist three varied feeder-free layer culture systems. The ES D3 cells are passaged 10 times in vitro and their clone are observed morphologically. In order to identify ES cell pluripotent and undifferentiation on the above mentioned systems, we tested the alkaline phosphatase (AKP) staining which is a marker of the undifferentiated cells and the experiment of ES D3 cells differentiation in nude mice were investigated. Results On both feeder layer systems and feeder free systems, the typical ES cells clone shape revealed as follows: nested aggregation (clone) with clear edge and smooth surface as well as close arrangement within the clone. Strong positive AKP reactive cells are observed. On the other hand. the teratomas made up with various tissues are formed in nude mice. Conclusion Each of the six culture systems can be used to promote the growth of ES D3 and maintain their undifferentiation and pluripotentiaty, which lay a solid foundation for the application of ES cells.