简易的单个肿瘤干细胞分离及其培养方法  

A simple method for isolating and culturing single cancer stem cells

作　　者：邓永键[1,2] 蒋强[1,2] 于莉娜 王红霞[1,2] 胡纯婷[1,2] 刘超[1,2,3] 丁彦青 

机构地区：[1]南方医科大学南方医院病理科 [2]南方医科大学基础医学院病理学系,广东广州510515 [3]广东省人民医院病理科,广东广州510080

出　　处：《南方医科大学学报》2012年第6期802-806,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(81172381);广东省高等学校科技创新重点项目(cxzd1016)~~

摘　　要：目的建立一套简单有效的单个肿瘤干细胞分离及培养方法,提高研究的效率与质量。方法在火焰上轻烤拉伸后的毛细管,与无菌塑料管连接建立口控毛细管单细胞分离装置。分离单个SW480细胞无血清培养筛选出克隆球,CD133、CK7荧光标记鉴定;消化克隆球吸取单个肿瘤干细胞并移至石蜡油封闭的微滴及96孔板中培养对比观察。结果 SW480细胞克隆形成率约为1.04%,其CD133表达强CK7表达弱;口控毛细管单细胞分离装置分离单个肿瘤干细胞的有效率可高达98.99%;单个肿瘤干细胞在微滴中培养与在96孔板中培养的比较:在分裂时间点上,前两次分裂时间相差无几(P>0.05),在微滴中培养的细胞后续分裂需时较长;在持续扩增的数量上,微滴培养为11.5%(22/192)而96孔板为9.2%(17/184),两者差异无统计学意义(P>0.05)。结论单个SW480细胞无血清培养可形成肿瘤干细胞球,口控单细胞分离装置能够有效分离单个肿瘤干细胞,石蜡油封闭微滴培养法和96孔板培养法均能够有效扩增单个肿瘤干细胞。但石蜡油封闭微滴培养法操作灵活、观察便捷、环境稳定,可作为培养单个肿瘤干细胞的首选。Objective To develop an effective method for isolating and culturing single cancer stem cells. Methods The capillary glass tube was stretched on fire and connected to a sterile plastic tube to prepare the single cell separation apparatus. Single SW480 cell clone spheres in serum-free culture were marked with CD133 and CK7, and the single cancer stem cells were separated and cultivated in 96-well plates or microdrops covered by paraffin. Results SW480 cell clone formation rate was about 1.04%, and the cell done spheres highly expressed CD133 with low CK7 expression. The isolation of the single cancer stem cells showed a success rate of 98.99% using the separation device. The cell division profile was comparable between the cell cultures in microdrop and 96-well plates in the initial 2 cell divisions （P〉0.05）, whereas prolonged cell division occurred afterwards in the microdrop culture as compared to 96-well plate culture. The cell population expansion of the single cancer stem cells was similar between microdrop culture （11.5%, 22/192） and 96-well plate culture （9.2%, 17/184） （P〉0.05）. Conclusion Single SW480 cells can develop into cancer stem cell spheres. Microdrop culture is convenient and stable, and can be the primary choice for single cancer stem cell culture.

关 键 词：肿瘤干细胞 单细胞分离 微滴培养 

分 类 号：R730.2[医药卫生—肿瘤]