Pig Bladder Smooth Muscle Cells And Transitional Epithelial Cells In Vitro And Bracket Complexes

Part I Primary cultivation and identification of smooth muscle cells in the bladder of diannan pigsObjective:To investigate the methods of extracting materials, isolation in vitro, primary cultivation, amplification and identification, and provide experimental evidence for the role of seed cells in tissue engineering.Methods:We performed the surgical operation to take bladder tissues of diannan pigs with small ears under the sterile condition. After the process of mechanical disintegration, we harvested bladder smooth muscle cells with a mixed enzyme method. DMEM/F12 was used as basal medium, and we cultivate the cells in 10% fetal calf serum. Morphological changes, growth and proliferation of cells were observed. We used the method of MTT to analyze the cell growth curve. Smooth muscle cells were identified by the methods of H-E staining, immunohistochemistry staining and electron microscope.Results:Cells in vitro can be integrated into chip and generally elongated and spindle shaped under inverted phase contrast microscope, showing a typical "valley and peak" form. The growth curve suggesedt the cell growth and proliferation in a good condition. Hematoxylin-eosin staining indicated these cells were consistent with smooth muscle cells. Smooth muscle actin staining was positive, and these cells were consistent with features of muscle cells under the transmission electron microscope.Conclusion:This method is simple and easy to be used for cultivating a large number of high-purity pig bladder smooth muscle cells. We constructed a stable culture system. In addition, the high-purity pig bladder smooth muscle cells can be used as seed cells in tissue engineering.PartⅡPrimary culture and identification of transitional epithelial cell in the diannan pig bladderObjective:To investigate the methods of isolation, cultivation, amplification and dentification. To provide an experimental evidence for the role of urinary in tissue engineering.Methods:We performed the surgical operation to take bladder tissues of diannan pigs with small ears under the sterile condition and put the tissue in neutral protease. After the process of peeling tissues carefully, we harvested high purity urinary bladder epithelial cells with a low concentration of trypsin digestion. We cultivate the cells in DK-SFM(With epidermal growth factor, insulin). Morphological changes, growth and proliferation of cells were observed. We used the method of MTT to analyze the cell growth curve. Smooth muscle cells were identified by the methods of MTT, H-E staining and immunohistochemistry staining.Results:A large number of high purity urinary bladder epithelial cells with a good activity can be harvested with this method. A large number of primary culture cells became adherent and had morphological changes after 16 hours.4 to 5 days later, primary cells can be fused to the 80%-90%, showing a typical "cobblestone"-like structure. Cells can grow stable after passage. No pollution hybrid cell was observed under the microscope. After passage, the cell growth accelerated. Cells can passage about 3-4 days and have regular shape before the 6 passages.Conclusion:With the combined method of cold neutral protease digestion overnight, low concentration of trypsin heat digestion, as well as primary and passaged cell culture, we can harvest high-purity transitional epithelial cell with a good cell viability, which can provide experimental evidence for these cells to be used as seed cells in tissue engineering of urinary.PartⅢConstruction of smooth muscle cells, transitional epithelial cells of pig bladders and SIS complexObjective:To observe the growth of smooth muscle cell and transitional epithelial cell of pig bladders in small intestinal submucosa, and provide experimental evidence of the two kinds of cells and SIS as urinary organs with the construction of tissue engineering scaffolds. Methods:We cultivated pig bladder smooth muscle cells and transitional epithelial cells in SIS, as well as cultivated the two different kinds of cells in SIS complex for 1 week and 10 days. Then, we observed growth of the two kinds of cells in SIS and SIS complex with the methods of specimens being fixed in 4% paraformaldehyde, embedded in paraffin, serially sectioned and stained with hematoxylin eosin and scanning electron microscope.Results:According to HE stained paraffin sections and scanning electron microscope, we found that there was fiber mesh structure in SIS. Bladder smooth muscle cells and transitional epithelial cells can grow in SIS with good morphous. Cells in SIS complex can grow in two sides and the growth of cell fusion was observed.Conclusion:Bladder smooth muscle cells and transitional epithelial cells can grow in SIS with good morphous and viability. SIS can provide a three-dimension for the attachment, growth and metabolism of bladder smooth muscle cells and transitional epithelial cells. This is an appropriate scaffold for tissue engineering of urinary.