Effects Of Conditioned Medium From Mammary Epithelial Cell On Proliferation And Epithelial Cell Differentiation Of Human Adipose-derived Stem Cells

Part I Isolation and culture of human mammary epithelial cells and preparation of conditioned mediumObjective To isolate and culture mammary epithelial cells of human breast hypertrophy, and prepare the conditioned mediums from mammary epithelial cell.Methods Mammary epithelial cells of human breast hypertrophy were isolated and cultured by the collagenase digestion method, and were subcultured to the third passage. The cryopreserved human normal breast epithelial cell strain (HBL-100) were resuscitated and cultured. Cell proliferation was tested by MTT method. Conditioned mediums from two different mammary epithelial cells were prepared, and their quality testing were done finally.Results Two cultured cells growed well, and they had typical epithelial appearance. The optical density (OD) value by MTT method showed a significant difference between two cultured cells (P<0.05).The acquired conditioned mediums had clear appearance, they were sterile, their pH value was7.35±0.06,and the glucose content was100mg/dl.Conclusion Under the same culture conditions, the proliferation ability of HBL-100is higher than that of mammary epithelial cells of human breast hypertrophy.The conditioned mediums from two different mammary epithelial cells were prepared successfully, and they meet the basic conditions of cell culture. Part Ⅱ Effects of two different conditioned mediums on proliferation of human adipose-derived stem cellsObjective To investigate the difference between two different conditioned mediums on growth and proliferation of human adipose-derived stem cells (ADSCs), in order to select suitable conditioned medium for epithelial cell differentiation of ADSCs.Methods ADSCs were isolated and cultured in vitro, and expression of CD13、CD29、 CD44、CD45、CD90and CD105in ADSCs were detected by flow cytometry. ADSCs were induced into lipocyte and chondrocyte in vitro, respectively. ADSCs were cultured by two different conditioned mediums, the growth process of ADSCs were observed under the inverted contrast microscope, and effects of two different conditioned mediums on proliferation of ADSCs were investigated by MTT method.Results The cultured human ADSCs growed well with typical fibroblast appearance. Expression of CD13、CD2、CD44、CD90and CD105were positive, and expression of CD45was negative by flow cytometry. The induced cells were presented the phenotypic characteristics of the lipocyte and chondrocyte. The number of cell decreased after ADSCs were cultured by conditioned medium from mammary epithelial cells of human breast hypertrophy, however, the cell proliferated well after ADSCs were cultured by conditioned medium from HBL-100.Conclusion The conditioned medium from human normal breast epithelial cell strain (HBL-100) is suitable for culturing ADSCs, but the conditioned medium from mammary epithelial cells of human breast hypertrophy is not suitable for culturing ADSCs. Part Ⅲ Conditioned medium induced differentiation of human ADSCs into mammary epithelial cellObjective To evaluate the differentiation potential of human adipose-derived stem cells(ADSCs) into mammary epithelial cell by conditioned medium.Methods ADSCs were cultured by conditioned medium in experimental group and DMEM/F12+10%FBS in control group for16days, respectively. The zona occludens protein1(ZO1)and cytokeratin18(CK18) protein expression in every group were detected by Western blotting. The induced cells were cultured in3D Matrigel for16days, expression of anti-collagen IV were detected by immunofluorescence cell staining, and the a-casein mRNA of every group were detected by QRT-PCR.Results After14days, the induced cells changed their morphology into epithelial-like shape. Compared with control group, the expression of ZO1and CK18protein in experimental group increased significantly,which was showed by Western blotting (P<0.05). After16days, these cells cultured in3D Matrigel formed cubic mass-like structure, which is similar to the normal acinar and ductal structure in vivo.Positive expression of anti-collagen IV were detected by immunofluorescence cell staining, and the QRT-PCR showed the a-casein mRNA expression in experimental group were enhanced.Conclusion ADSCs could be induced and differentiated into mammary epithelial cell by conditioned medium from human normal breast epithelial cell strain (HBL-100), providing a new cell resource for breast tissue engineering.