ObjectiveTo isolate cancer stem cells (CST) from human breast cancer cell line (MCF-7) and study their sensitivity toward oxidative stress.MethodsMCF-7 cells were cultured in serum-free suspension culture medium to identify cells forming the sphere phenotype. The morphological changes of MCF-7 cells were observed by inverted phase contrast microscope (compared with MCF-7 cells cultured in serum-free suspension culture medium). The expression of CST marker CD133 was detected by immunocytochemical staining in CST cell spheres (experimental group) with a diameter of 100 μm and MCF-7 cells (control group) with a fusion degree of 70%. The positive rate of CD133 was detected by flow cytometry in the third generation of tumor cells with diameter of 150 μm. The second generation of tumor globular cells (experimental group) with diameter of 150 μm and corresponding MCF-7 cells (control group) were taken to be damaged by 50 mol/L H2O2 for 120 minutes. The expression of DNA damage marker histone H2AX phosphorylation (γH2AX) was detected by immunocytochemical staining.ResultsInverted phase contrast microscopy showed that MCF-7 cells grew initially in a single-cell adherent state, then aggregated and grew in serum-free suspension culture medium, and finally formed CST cell spheres, while the control MCF-7 cells cultured in MCF-7 cell culture medium grew extensively and could not grow in suspension. Fluorescence microscopy showed that the expression of CD133 in MCF-7 cells of control group was negative, while that in experimental group was positive. Flow cytometry showed that CD133 was positive in CST cells, and the positive rate was 92%. Inverted fluorescence microscopy showed that the expression of γH2AX in CST tumor spheres of experimental group was significantly lower than that in MCF-7 cells of control group after 120 minutes of H2O2 injury.ConclusionSerum-free suspension culture medium can produce globular CST cells from MCF-7 tumor cell line, which have strong antioxidant damage.