心脏原位干细胞(cardiac stem cells,CSCs)治疗心肌梗死具有公认的疗效,但是其分离及培养技术尚不完善,这是制约其临床应用的关键技术问题。为解决这一问题,本研究旨在优化Lin~-(lineage-negative)Sca-1~+(stem cell antigen-1-positive)CSCs分离方案。用组织块酶解法(混合酶液)分离C57BL/6J新生(出生0～3天)小鼠Lin~- Sca-1~+ CSCs。在已有的研究基础上,严格控制消化时间、次数、温度、搅拌速度、离心时间和转速等多重因素,结合免疫磁珠分选获得纯度较高的Lin~- Sca-1~+ CSCs。此后,对分离纯化的原代细胞进行培养,优化培养基成分、换液时间和方式。采用流式细胞术及免疫荧光染色技术,检测分选细胞的纯度及传代培养细胞中Sca-1~+细胞的百分比。结果显示:(1)本法分离获得的Lin~- Sca-1~+ CSCs纯度高达(85.03±5.60)%;(2)离体培养过程中,细胞生长状态良好,原代培养5天开始有干细胞克隆球生成,培养7天即可铺满皿底,生长曲线显示,离体培养第3天细胞进入对数增长期;(3)免疫组化染色结果显示:干细胞特异性标志Sca-1的表达随着传代的进行有一定的衰减。流式细胞术检测结果显示,第一代、第三代和第五代培养细胞的Sca-1阳性率分别为(71.82±2.63)%、(58.38±3.70)%、(46.19±4.72)%。以上结果表明,本研究所建立的组织块酶解法结合免疫磁珠分选法可获得纯度较高的Lin~- Sca-1~+ CSCs,同时干细胞离体培养体系相对稳定。本研究所建立的分离及培养方法简单稳定、可靠有效,为进一步研究Sca-1~+ CSCs治疗心肌梗死奠定了良好的方法学基础。 Cardiac stem cells (CSCs) have a proven efficacy in the treatment of myocardial infarction, but their isolation and culture techniques are still not perfect, which is a key technical issue restricting their clinical application. In order to solve this problem, this study aimed to optimize the lineage-negative Sca-1 ~ + stem cell antigen-1-positive CSCs isolation scheme. C57BL / 6J neonatal (born 0-3 days) mice Lin ~ - Sca-1 ~ + CSCs were isolated by tissue block enzymatic method (mixed enzyme solution). Based on the existing research, we strictly control the digestion time, the number of times, the temperature, the stirring speed, the centrifugation time and the rotational speed and so on. The Lin (superscript -) Sca-1 + CSCs with high purity were obtained by immunomagnetic bead sorting. Thereafter, the primary cells isolated and purified are cultured, the medium components are optimized, and the time and manner of liquid exchange are optimized. The purity of sorted cells and the percentage of Sca-1 ~ + cells in subcultured cells were detected by flow cytometry and immunofluorescence staining. The results showed that: (1) The purity of Lin ~ - Sca-1 ~ + CSCs obtained by this method was as high as (85.03 ± 5.60)%; (2) During the in vitro culture, the cell growth was good, Stem cell clone spheres were generated, and the bottom of the dish was covered after 7 days of culture. The growth curve showed that the cells in logarithmic growth phase were in vitro cultured on the 3rd day. (3) The results of immunohistochemical staining showed that the stem cell-specific marker Sca-1 The expression decays with the passage of time. Flow cytometry results showed that the positive rates of Sca-1 in the first, third and fifth generation cultured cells were (71.82 ± 2.63)%, (58.38 ± 3.70)% and (46.19 ± 4.72)%, respectively. The above results showed that Lin ~ - Sca-1 ~ + CSCs with high purity could be obtained by tissue enzymolysis combined with magnetic bead sorting method in this study. Meanwhile, the system of stem cells cultured in vitro was relatively stable. The method of isolation and culture established in this study is simple and stable, reliable and effective, which laid a good methodological foundation for further study on the treatment of myocardial infarction by Sca-1 ~ + CSCs.