简易的单个肿瘤干细胞分离及其培养方法

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。

A simple method for isolating and culturing single cancer stem cells

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 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.