In Vitro Culture And Identification Of Rabbit Tracheal Epithelial Cells And Bone Marrow Mesenchymal Stem Cells

ObjectiveTo investigate in vitro culture techniques and identification methods of rabbit tracheal epithelial cells and bone marrow mesenchymal stem cells.1. To establish the model of culture and identification of tracheal epithelial cells in vitro, to explore their regular patterns in the fields of growth and passage.2. To construct tracheal epithelial cell sheets in vitro.3. To establish the model of isolation and culture BMMSCs in vitro, which may provide experimental methods and technical supports for the study in the differentiation from BMMSCs to the epithelial cells.Methods1. In vitro epithelial cells culture1) Rabbit tracheal epithelial cells would be obtained by tissue-explant method and two-step enzyme digestion method and then be passaged.2) The3rd generation epithelial cells, which had been purified, and cryopreservating ones would be planted into96-well plates respectively, and then be examined the cell proliferating activity at6,12,24,36,48,60,72hours later by MTT method.3) After purification, the form and structure of the cultured epithelium would be observed by HE staining and environment scanning electron microscope (ESEM), protein expression (PCK/CK-18) in the epithelial cells would be examined by immunocytochemistry with SABC method and immunofluorescence test with FITC labeled fluorescent antibody.2. In vitro epithelial cell sheets constructionPurified epithelial cells, which were obtained from tissue-explant method, would be used as seed cells to plant into the temperature-responsive culture dishes. The epithelial cell sheets would also be identified by HE staining, immunocytochemistry and immunofluorescence methods, ESEM.3. In vitro BMMSCs cultureBMMSCs, which would be isolated by the cell density gradient centrifugation method and the whole blood culture method, would be cultured and passaged. The antigen markers on the surface of these cells would be identified by FCM.Results1. In vitro epithelial cells culture1) Rabbit tracheal mucosal epithelial cells could be obtained by tissue-explant method and two-step enzyme digestion method. Cells from tissue-explant method were less damag ed during the isolating period, containing more hybrid cells, but could be purified by cell-purification treatment. Mechanical curettage and other cell-purification methods contributed to the further purification of the epithelial cells.2) After resuscitation, cells, which were frozen three months, had high survival and adherence rate, but the rate of growth and proliferation was obviously lower than the unfrozen cells.3) HE staining showed that the cultured cells were spindle-shaped, round, polygon and et al. The nuclei were pale blue and cytoplasm was pale red.4) Immunocytochemistry test with SABC method showed that cytoplasm was tan and nuclei were pale blue in PCK and CK-18group, but cytoplasm was not tan in control group, which demonstrated that cultured cells were epithelial cells.5) Immunofluorescence staining with FITC labeled fluorescent antibody show that the shape of cells and nuclei were visible and cytoplasm was light green in PCK and CK-18group, but cytoplasm was not light green in control group, which demonstrated that cultured cells were epithelial cells.6) EMSM testing showed that a part of cells had cilia, which demonstrated that cultured cells were epithelial cells.2. In vitro epithelial cell sheets construction1) The purified epithelial cells, which were obtained from tissue explant method and used as seed cells, were planted into the temperature-responsive culture dishes. Ten to fourteen days later, epithelial cells coverd the whole bottom of the dishes. The day after changing the medium, we could harvest epithelial cell sheets.2) HE staining showed that cells arranged closely, the nuclei were pale blue and cytoplasm was pale red.3) Immunocytochemistry test with SABC method showed that cells arranged closely, cytoplasm was tan and nuclei was pale blue in PCK and CK-18group, but cytoplasm was not tan in control group, which demonstrated that constructed cell sheets were epithelial cell sheets.4) Immunofluorescence staining with FITC labeled fluorescent antibody show that cells arranged closely, the shape of cells and nuclei were visible and cytoplasm was light green in PCK and CK-18group, but cytoplasm was not light green in control group, which demonstrated that constructed cell sheets were epithelial cell sheets.5) EMSM testing showed that cells arranged closely, cilia could be seen on the surface of a part of cultured cells, which demonstrated that constructed cell sheets were epithelial cell sheets.3. In vitro BMMSCs culture1) Whole blood culture method and cell density gradient centrifugation method could be used to obtain BMMSCs. The cells were spindle, round, irregular-shaped, et al.2) FCM was used to identify the surface antigen markers of BMMSCs, high expression of CD29and CD44, no expression of CD14and CD34.Conclusion1. Tissue-explant method and two-step enzyme digestion method were successfully used to construct primary culture models of tracheal epithelial cells in vitro.2. Tracheal epithelial cell sheets were successfully constructed in vitro by using temperature-responsive culture dishes.3. Rabbit BMMSCs were successfully isolated and cultured in vitro. In addition, the cell surface antigen markers were also identified successfully. These could provide theoretical basis and technical support for the further study in differentiation from HBMMSCs to epithelial cells.