A noninvasive transfer system for polarized renal tubule epithelial cell sheets using temperature-responsive culture dishes

Abstract

We used temperature-responsive culture dishes onto which the temperature-responsive polymer, poly(Nisopropylacrylamide), was covalently grafted for tissue engineering. Confluent cells harvested as intact sheets from these surfaces by simple temperature reduction can be transferred to various surfaces including additional culture dishes, other cell sheets, and tissues. In order to examine the maintenance of cell polarity, Madin-Darby canine kidney cells and human primary renal proximal tubule epithelial cells which had developed apical-basal cell polarity in culture, were subjected to cell sheet transfer. This functional and structural cell polarity, which is susceptible to treatment with trypsin, was examined by immunohistochemistry and transmission electron microscopy. Using our cell-sheet method, the noninvasive transfer of these cell sheets retaining typical distributions of Na+/K+-ATPase, GLUT-1, SGLT-1, aquaporin-1, neutral endopeptidase and dipeptidylendopeptidase IV, could be achieved. The transferred cell sheets also developed numerous microvilli and tight junctions at the apical and lateral membranes, respectively. For biochemical analysis, immunoblotting of occludin, a transmembrane protein that composes tight junctions, was conducted and results confirmed that occludin remained intact after cell sheet transfer. This two-dimensional cell sheet manipulation method promises to be useful for tissue engineering as well as in the investigation of epithelial cell polarity.