Shiroyanagi Yoshiyuki, Yamato Masayuki, Yamazaki Yuichiro, Toma Hiroshi, Okano Teruo
Institute of Advanced Biomedical Engineering and Science and Department of Urology, Tokyo Women's Medical University, Tokyo, Japan.
Tissue Eng. 2003 Oct;9(5):1005-12. doi: 10.1089/107632703322495646.
Augmentation cystoplasty using gastrointestinal flaps may induce severe complications such as lithiasis, urinary tract infection, and electrolyte imbalance. The use of viable, contiguous urothelial cell sheets cultured in vitro should enable us to avoid these complications. Transplantable urothelial cell sheets were obtained by utilizing a temperature-responsive cell culture method, and then examined by immunostaining and electron microscopy. Canine urothelium was produced on the surfaces of temperature-responsive culture dishes covalently bonded with the thermally sensitive polymer, poly(N-isopropylacrylamide). Stratified urothelial cell sheets were cultured and then harvested intact without enzymatic treatment from these dishes by reducing the temperature. Histological structure and cell-to-cell junctions were compared between these urothelial cell sheets and those harvested with dispase. All urothelial cell sheets were harvested from the bonded surfaces by reducing the culture temperature without the need for dispase. Electron microscopy revealed well-developed microridge, microvilli, and cell junction complexes. Conversely, these same cell features were destroyed by dispase treatment. Immunoblotting revealed that dispase fragmented occludin, whereas it remained unchanged in the intact urothelial cell sheets. Novel urothelial cell sheets obtained by culture on temperature-responsive culture surfaces were successfully harvested much less destructively than with dispase. This technology should prove useful in urinary tract tissue engineering in the near future.
使用胃肠道皮瓣进行膀胱扩大术可能会引发严重并发症，如结石形成、尿路感染和电解质失衡。使用体外培养的有活力的、连续的尿路上皮细胞片应该能够帮助我们避免这些并发症。通过利用温度响应性细胞培养方法获得可移植的尿路上皮细胞片，然后通过免疫染色和电子显微镜进行检查。犬尿路上皮在与热敏聚合物聚（N-异丙基丙烯酰胺）共价结合的温度响应性培养皿表面产生。培养分层的尿路上皮细胞片，然后通过降低温度从这些培养皿中完整收获而无需酶处理。比较这些尿路上皮细胞片与用dispase收获的细胞片之间的组织结构和细胞间连接。通过降低培养温度从结合表面收获所有尿路上皮细胞片，无需dispase。电子显微镜显示微嵴、微绒毛和细胞连接复合体发育良好。相反，这些相同的细胞特征在dispase处理后被破坏。免疫印迹显示dispase使闭合蛋白断裂，而在完整的尿路上皮细胞片中其保持不变。通过在温度响应性培养表面上培养获得的新型尿路上皮细胞片成功收获，其破坏性比用dispase收获的要小得多。这项技术在不久的将来应该会在尿路组织工程中证明是有用的。
