Preliminary Study On The Preparation Of Tissue Engineered Urinary Tract Cell-scaffold Composite And Cutivation Of It In Vitro

Tissue engineering and regenerative medicine are the most promising techniques for treating the failure and decline of tissues and organs. Tissue engineering in urologic system has experienced rapid improvements since the rise. Overseas scholars have already developed tissue engineered bladders, ureters and urethra which proved useful for relevant tissue replacement. Because the complicated micro-environment for normal UEC and urinary SMC growing is not easy to duplicate in vitro, cultivation of UEC and urinary SMC are really tough work for many domestic tissue engineering labs, and as a matter of fact, the technique for expanding them in vitro plays a key role in urinary tissue engineering. In this study, we managed to explore the easier and convenient methods for proliferation UEC and urinary SMC to get enough cells for tissue engineering urinary organs in a short time. In addition, the cells can be used for other fields which are based on these kinds of cells.The study comprises of three parts. The first part is concerned of cultivation of human normal UEC . The tissue samples were harvested on surgery of the patients who suffered from inherent ureteropelvic junction obstruction, renal cell carcinoma, benign prostate hyperplasia, and it has been proved that no cancer from transitional cells by pathological examination. We compared the two culture systems for UEC growing, one is K-SFM added with BPE and rEGF, the other isRPMI1640 added with FBS. When cells grew we found that RPMI1640 system cannot inhibit fibroblast and SMC growth, but cells growed in the K-SFM system contained mainly UEC without frbroblasts and SMC. UEC climbed out of the explants and grew gradually to a monolayer for about 12 to 14 days. Immunocytochemistry coloration for pancytokeratin of the cells got positive result. Moreover transmission electric microscopy examination of the cells confirmed the derivation of UEC. After that, we observed the viability of the UEC obtained, and the cell growth curve and attachment efficiency showed good activities. Furthermore, the UEC was cryopreserved in liquid nitrogen for about 3 months and they gained the viability when they were thawed. All the results indicated that the K-SFM is an appropriate culture system for UEC growth and enough UEC can be produced in this way.In the second part of the study, we primarily cultivated human bladder, ureter SMC under the condition of DMEM plus FBS and antibiotics. 20 samples were harvested according to aseptic principles. All the samples were cut into 3 parts, one part was digested by 0. 2% I collagenase, and we failed to get seprate cells, one part was digested by compound enzyme of 0. 2% I collagenase and 2. 5mg/ml dispase and 3% FBS, enough separate cells were harvested and were seeded. The other part was prepared for explant culture, when cells grew, we recorded the differences of success rates and first subculture interval between the two groups. In the end, it was clear that explant cell culture was superior to the enzymatic digestion cell culture in success rate. In addition, the latter one cost lesser time for the first subculture. In order to identify the cells derivation, immumocytochemistry for anti a -actin of the cells was performed and positive result was received which made sure that the cell came from USMC. Then the cell growth curve, attachment efficiency we achieved demonstrated USMC were of normal vitality. We also cryopreserved some USMC and they recovered to the...