Background and objective: There are no universal method to recognize and screen for lung cancer stem cell markers and indicators. Commonly used methods are flow Cytometry and learning from other cancer stem cell sorting tags to sort lung cancer stem cells. But this method has low specificity screening, the workload is huge. In this study, Serum-free suspension culture was used to enrich lung cancer stem cells, and explore method for lung cancer stem cell screening.
Methods: Human large lung cancer cell line-L9981 was cultured in serum-free and growth factors added medium, and spheres were obtained. Then the morphological differences of sphere cells and adherent L9981 cells cultured in serum-containing mediums are observed. Cell proliferation was analyzed by Vi-cell viability analyzer; invasion ability was tested by transwell assay; and in vivo tumorigenicity of the two groups of cells was studied in nude mouse.
Results: Compared with adherent L9981 cells cultured in serum-containing mediums, cells cultured in serum-free medium display sphere appearance. Doubling time of adherent cells and sphere cells are (56.05±1.95) h and (33.00±1.44) h respectively; Spheroid cells had higher invasion and tumorigenicity ability, 5 times and 20 times respectively, than adherent cells.
Conclusion: Suspension cultured L9981 in Serum-free medium could form spheroid populations. Cells in spheres had higher ability of invasion and Tumorigenicity than adherent L9981 cells. These results indicated spheroid L9981 cells contained enriched lung cancer stem cells, and Serum-free suspension culture can be a candidate method for enriching lung cancer stem cell.
背景与目的: 目前国内外还没有确切的、得到公认的肺癌干细胞的筛选标记分子、指标和方法，常用方法为通过流式细胞技术，借鉴其他肿瘤干细胞分选标记来分选肺癌干细胞，但其筛选特异性低、工作量巨大。本研究采用无血清悬浮培养法富集肺癌干细胞，对肺癌干细胞的筛选方法进行探索。
方法: 采用添加生长因子的无血清培养基对人大细胞肺癌细胞株L9981进行悬浮培养，获得肺癌细胞球体。对含血清培养贴壁L9981细胞和无血清培养成球后的L9981细胞，通过显微镜下观察比较二者的生物学形态，应用Vi-cell细胞活力分析仪计数细胞并绘制生长曲线比较二者的增殖能力，通过Transwell实验研究它们的侵袭能力差异，并通过接种裸鼠观察二者在体内的成瘤性来研究肺癌细胞球体的生物学功能。
结果: 与血清贴壁培养L9981细胞相比，无血清培养L9981细胞成球形生长，贴壁L9981和L9981球体细胞的倍增时间分别为（56.05±1.95）h和（33.00±1.44）h，球体细胞的侵袭和成瘤能力分别为贴壁L9981细胞的5倍和20倍。
结论: 通过无血清悬浮培养的L9981细胞可以形成肺癌球体细胞群，L9981球体细胞的侵袭和成瘤能力均明显高于贴壁L9981细胞，显示L9981球体细胞中富集了肺癌干细胞样的细胞。无血清悬浮培养肺癌球体细胞可作为富集肺癌干细胞样细胞的一种候选方法。