Development of Cultured Renal Tubules with Cell Sheet Engineering

通过共价接枝聚N-异丙基丙烯酰胺，使其表面性质发生亲水/疏水的变化，研制了一种温度响应型培养皿。该表面显示出类似于商业组织培养聚苯乙烯皿的弱疏水性，并且在37℃下允许各种细胞在其上粘附和铺展。但当温度降低到32℃以下时，细胞从高度水合的表面脱离，而不需要任何蛋白水解酶如胰蛋白酶。所有通过细胞-细胞连接点连接的铺展细胞作为单个连续细胞片收获。在本研究中，我们研究了利用收获的肾上皮细胞片在生物人工肾小管的发展，结合细胞片和多孔膜透析的分子运输和生产。我们开发了一种从培养皿到其他表面（如多孔膜）的非侵入性细胞片操作方法。将原代人肾上皮细胞与多孔膜结合制备实验装置。各种细胞表面蛋白的表达和定位通过免疫染色和共聚焦激光扫描显微镜证实。

We developed a temperature-responsive cultured dishes by covalent grafting of poly (N-isopropylacrylamide) which shows the hydrophilic/hydrophobic change of surface property. This surface shows weak hydrophobicity similarly to commercial tissue culture polystyrene dishes and allows various cell adhesion and spreading on it at 37℃. But the cells are detached from the highly hydrated surfaces without any need of proteolytic enzymes such as trypsin after reducing temperature below 32℃. All the spread cells connecting via cell-cell junctions are harvested as a single contiguous cell sheet. In the present study, we examined the utilization of harvested kidney epithelial cell sheets in the development of bioartificail renal tubules by combining the cell sheets and porous membranes for dialysis in terms of molecular transport and production. We developed a non-invasive cell sheet manipulation method from culture dishes to other surfaces such as porous membranes. Experimental devices were prepared by combining of primary human kidney epithelial cells and porous membranes. The expression and localization of various cell surface proteins are confirmed by immunostaining and confocal laser scanning microscopy.