成人心肌干细胞的体外培养纯化与鉴定

Isolation,in vitro Culture and Identification of Cardiac Stem Cells from Adult Human Heart

目的:自成人心脏组织培养获得的细胞中分选得到心肌干细胞,体外培养并鉴定其表面标记。方法:手术中取下的非心肌病患者心脏组织标本,通过组织块培养法及冲洗获得的细胞,以免疫磁珠进行分选,纯化后的细胞在条件培养基中培养,待增殖至一定数量后进行表面标记的流式细胞鉴定。结果:心肌组织块培养可获得小、圆、亮细胞,将其冲洗下来后以c-kit磁珠分选,之后在通过胎鼠成纤维细胞制备的条件培养基中培养,可增殖并形成心球样细胞团结构,流式细胞仪鉴定其为c-kit+。结论:通过本实验的方法可以在成人心肌组织中分离并纯化得到c-kit+的心肌干细胞,其可在体外增殖形成典型的心球样细胞结构并可保持表面的c-kit干细胞标记。

Objective：To isolate and culture cardiac stem cells （CSCs） in vitro and evaluate their characterization. Methods：Myocardial tissues obtained from adult human heart were minced into pieces of 0.5 mm3 to 1.0mm^3, and digested thrice for 5 min at 37 ℃ with 0.2% trypsin and 0.1% collagenase IV. The remaining tissue fragments were cultured in complete explants medium （CEM） at 37 ℃ in the presence of 5% CO2. About 3 to 4 weeks later, a layer of fibroblast-like cells were generated from the adherent explants over which small, round, phase-bright cells migrated. Collected these phase-bright cells by washing with D-Hanks and sorted with anti-c-kit-microbeads. Isolated cells were cultured with conditioned cardiosphere-growing medium （CCGM） and evaluated with flow cytometry. Results：Sorted phase-bright cells via MACS were cultured in CCGM. After 10 days the adherent cells sprout new round-bright cells at their cell body and form three-dimensional spheres. Flow cytometry demonstrated the phenotype of cells were c-kit^＋. Conclusion：A c-kit^＋ cell population has been obtained from adult human heart, which can form cardiospheres and can maintain the characterization of stem cells（c-kit^＋） in vitro culture.