Pokrywczynska Marta, Czapiewska Monika, Jundzill Arkadiusz, Bodnar Magdalena, Balcerczyk Daria, Kloskowski Tomasz, Nowacki Maciej, Marszalek Andrzej, Drewa Tomasz
Chair of Urology, Department of Regenerative Medicine, Ludwik Rydygier Medical College in Bydgoszcz, Nicolaus Copernicus University in Torun, Karlowicza 24 Street, Bydgoszcz, 85-092, Poland.
Department of Clinical Pathomorphology, Ludwik Rydygier Medical College in Bydgoszcz, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland.
Cell Biol Int. 2016 Jul;40(7):812-20. doi: 10.1002/cbin.10614. Epub 2016 May 20.
Many experimental approaches have been conducted in order to isolate urothelial cells from bladder tissue biopsies, but each method described has utilized different protocols and sources of bladder tissue. In this study, we compared the different methods of urothelial cell isolation available in literature together with standardized methods in order to obtain more unified results. Five methods for primary porcine urothelial culture establishment were compared: tissue explants and four enzymatic methods utilizing collagenase II, dispase II, combination of dispase II and trypsin, and trypsin alone. The average number of isolated cells, cell morphology, success of established culture, average number of cells from the first passage, expression of p63 and pancytokeratin and the characterization of urothelial cell growth, and aging were analyzed during the in vitro culture. The method utilizing dispase II was the most efficient and reproducible method for the isolation and culture of porcine urothelial cells when compared to the other tested methods. Urothelial cells obtained by this method grew considerably well and the cultures were established with high efficiency, which enabled us in obtaining a large quantity of cells with normal morphology. Contamination with fibroblasts in this method was the lowest. The utilization of a proper method for urothelial cell isolation is a critical step in the urinary tract regeneration when using tissue engineering techniques. In summary, this study demonstrated that by utilizing the described method with dispase II, a suitable number of cells was achieved, proving the method useful for tissue regeneration.
为了从膀胱组织活检中分离尿路上皮细胞，人们已经进行了许多实验方法，但所描述的每种方法都采用了不同的方案和膀胱组织来源。在本研究中，我们将文献中可用的不同尿路上皮细胞分离方法与标准化方法进行了比较，以获得更统一的结果。比较了五种建立原代猪尿路上皮细胞培养的方法：组织块培养法和四种酶解法，分别使用胶原酶II、分散酶II、分散酶II与胰蛋白酶的组合以及单独使用胰蛋白酶。在体外培养过程中，分析了分离细胞的平均数量、细胞形态、培养建立的成功率、第一代细胞的平均数量、p63和全细胞角蛋白的表达以及尿路上皮细胞生长和衰老的特征。与其他测试方法相比，使用分散酶II的方法是分离和培养猪尿路上皮细胞最有效且可重复的方法。通过这种方法获得的尿路上皮细胞生长良好，培养建立效率高，这使我们能够获得大量形态正常的细胞。这种方法中纤维母细胞的污染最低。在使用组织工程技术进行尿路再生时，采用合适的尿路上皮细胞分离方法是关键步骤。总之，本研究表明，通过使用所描述的分散酶II方法，可以获得合适数量的细胞，证明该方法对组织再生有用。
