Two-dimensional renal cell-sheet manipulation for a novel bioartificial renal tubule

摘要

To develop a bioartificial renal tubule device, we have focused on a novel cell-sheet transfer method using temperature-responsive culture dishes and designated it 'two-dimensional (2-D) cell-sheet manipulation'. A temperature-responsive polymer, poly(iV-isopropy]acrylamide) is covalently grafted on tissue culture grade polystyrene (TCPS) dishes by radial polymerization initiated with electron-beam irradiation. This polymer grafted culture dish exhibits a temperature-responsive property; that is, the surface is slightly hydrophobic under the culture condition at 37(deg)C and reversibly changes to hydrophilic below the lower critical solution temperature. Renal tubule epithelial cells were cultured on the PIPAAm-grafted culture dishes. Cells attached, adhered, proliferated to confluency on these dishes as well as on TCPS dishes. After 3 weeks of culture, cells were subjected to the 2-D cell-sheet manipulation. Immunocytochemisty with anti-P-catenin antibody revealed that the cell-cell junctions were well organized in the 2-D manipulated cell sheets. The viability assay showed that the transferred cells were not damaged during the 2-D cell-sheet manipulation. Furthermore, the functional and structural cell polarity, which is susceptible to trypsin, was examined by confocal laser scanning microscopy and transmission electron microscopy. Renal epithelial cell sheets retained their apical-basal polarity even after the 2-D manipulation. Using temperature-responsive culture dishes, noninvasive cell-sheet manipulation was succeeded. These transferred renal tubule cell sheets promise to be useful for a novel bioartificial kidney and we believe that these 2-D cell-sheet manipulation techniques will become new revolutionary tools for tissue engineering.