Study Of Seed Cells For Tissue-engineered Trachea

Objective1. To establish a method for creating rabbit tracheal epithelial cell sheets in vitro, and explore their regular patterns in the fields of growth and passage for the provision of seed cells in tissue engineering of trachea.2. To explore and establish a method of isolation, culture, and identification of bone marrow mesenchymal stem cells (BMSCs) in vitro.3. By comparing the different separation methods of rabbit BMSCs, to find suitable BMSCs cultured ways for the provision of seed cells in tissue engineering of trachea.Methods1. The Creation of Rabbit Tissue Engineered Tracheal Epithelial Cell Sheets1) Rabbit tracheal epithelial cells would be obtained by tissue explant technique and then be passaged.2) Cytokeratin(CK-18) expressed in the epithelial cells would be examined by immunocytochemistry with SABC method and immunofluorescence test with FITC labeled fluorescent antibody.3) The second generation epithelial cells would be planted into96-well plates respectively, and then be examined the cell proliferating activity at6,12,24,36,48,60,72,84,96,108hours later by MTT method.4) The second generation epithelial cells would be passaged in UpCell culture dishes at37℃,5%CO2. About two days, cells have reached confluency, and tracheal epithelial cell sheet was prepared when the ambient temperature was changed to20℃, and then cell sheet is analyzed and identified. 2. Comparisons of rabbit bone marrow mesenchymal stem cell isolation and culture methods in vitro1) BMSCs were obtained by whole bone marrow adherent culture、half red blood cell lysis、 whole red blood cell lysis and Ficoll density gradient centrifugation.2) The ability of cell proliferation and expression of CD44, CD34cell marker were analyzed among the four uniquely isolated groups of BMSCs.3) Inverted phase contrast microscope was used to observe the morphological changes of cells. CD34, CD44antigens of BMSCs were identified by flow cytometry and immunity fluorescence. And Growth curves of the2nd,3rd,4th and5th generation BMSCs were drawn by MTT, too.Results1. The Creation of Rabbit Tissue Engineered Tracheal Epithelial Cell Sheets1) Epithelial cells were isolated from rabbit tracheal mucosa using tissue explant technique successfully, and all cells were positive for CK-18staining, showing they had properties of epithelial cells.2) The growth curves of the2nd passaged cells had an "S" shape and growing latent phases of passaged cells became stable at the6th to36th hour. The log phase of growth appeared about the36th to the96th hour and then cells reached the plateau.3) When the ambient temperature dropped at20℃, epithelial cells detached automatically from the UpCell culture dishes and formed an intact cell sheet. The cells are strongly attached and overlapping under light microscope.2. Comparisons of rabbit bone marrow mesenchymal stem cell isolation and culture methods in vitro1) BMSCs could be separated by four method successfully. Immunofluorescent staining indicated that the CD44-PE cell marker fluoresced green on the cell surface under a fluorescent microscope. CD34-PE was fluorescent brown (not green). So, cultured cells were positive for CD44, but negative for CD34.2) The method of untreated whole bone marrow adherent culture is superior to the other3 groups at the4th,7th,10th, and13th day (p<0.05), and there are no obvious differences among the four groups of BMSCs by flow cytometry analysis.3) The cells from the4th passage had the greatest proliferation ability (p<0.05), with a greater growth speed and cell quantity than other passages.Conclusion1) Tissue explant method was successfully used to construct primary culture models of rabbit tracheal epithelial cells in vitro. Rabbit tracheal epithelial cell sheet can be fabricated with UpCell culture dishes and it could provides a novel pathway for constructing tissue engineering trachea.2) BMSCs were separated by whole bone marrow adherent culture> half red blood cell lysis、 whole red blood cell lysis and Ficoll density gradient centrifugation successfully.3) The method of untreated whole bone marrow adherent culture is the best suitable cultured ways of rabbit BMSCs. The BM blood adherent culture technique did not affect BMSC heterogeneity, differentiation potential, or biological activity compared to the other methods.4) These techniques of rabbit tracheal epithelial cell sheet and BMSCs could laid a theoretical foundation of seed cells to construct tissue-engineered trachea, and make large-scale clinical application of tissue-engineered trachea become reality.