简易的单个肿瘤干细胞分离及其培养方法

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 clone spheres highly expressed CD133 with low CK7 expression.The isolation of the single canrer 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.