Isolation, Culture And Identification Of Human Breast Cancer Stem Cells

Objective:The separation,cultivation,and enrichment of tumor stem cells which is ready for later experiment from human breast cancer cells.Methods:We first make the HE staining and immunohistochemistry staining of tumor maker(ER PR HER-2 EGFR P53 Ki-67 Bcl-2 CK14) of this tissue. Then we separate the breast cancer primary cells by mechanical separation and enzyme digestion .Culturing these cells in serum medium and serum-free medium separately to make CA15-3 immunocytochemical stain of the former ones, and selecting monoplast clone by doubling dilution to analyze the condition of serum-free culture. We identify the cell-surface marker of the cultured cell-spheres and there biological character of proliferation and differentiation at last. In the experiment, we use MCF-7 cell line as control group.Results:The HE staining of this breast cancer tissue appeared tissue and cell heteromorphism. The result of immunohistochemistry staining is: ER(++),PR(-),HER-2(-),EGFR(-),P53(+),Ki-67(+),Bcl-2(++++)and CK14(-). The primary cells separated grow as flagstone; CA15-3 is espress on cell membrane and plasma. The phenotype of the human breast cancer stem cell is CD44+CD24-/low.Subculturing the cell sphere, we get more lager spheres. Culturing the cell sphere in serum medium, we get cell population of CD44+CD24+ and CD44-CD24+ by adherence. Conclusions:This breast cancer is infiltrating ductal carcinoma and we separate and culture the human breast cancer stem cells of CD44+CD24-/low which have the ability of proliferation and differentiation successfully. The cell surface marker CD44 and CD24 are changing during differentiation.