A Novel Transplantable Epidermal Cell Sheet Noninvasively Harvested from Thermoresponsive Cell Culture Dishes without Proteolytic Enzymes

为了开发未经疾病处理的可移植培养上皮的新技术，将人角质形成细胞镀在热响应性聚合物聚n -异丙基丙烯酰胺（poly（n -异丙基丙烯酰胺））的培养皿上。接枝的盘子表面在32℃以上为微疏水性，在32℃以下可逆地变为亲水性。根据Rheinwald和Green的方法，37℃下，与未移植的培养皿一样，移植表面角质形成细胞增殖并形成多层。多层角质细胞片只需要将温度降低到20℃即可从移植物表面分离，无需处理。培养皿上未观察到细胞残留物。在未移植的培养皿中未观察到这种细胞薄片脱离。采集的角化细胞薄片的免疫印迹显示，疾病处理破坏了e -钙粘蛋白和层粘连蛋白5，而这些分子在仅通过降低移植培养皿的温度采集的角化细胞薄片中保持完整。透射电镜显示，低温处理时桥粒被破坏，低温处理时桥粒被保留。研究了热反应培养皿作为组织工程的新工具，用于制备用于细胞移植的人造上皮以及研究完整的多层角化细胞片。经过无骨大鼠的移植实验，我们刚刚开始了组织工程表皮细胞片的临床应用。

To develop new technology for harvesting transplantable cultured epithelium without dispase treatment, human keratinocytes were plated on culture dishes grafted with a thermoresponsive polymer, poly(N-isopropylacrylamide). The grafted dish surfaces are slightly hydrophobic above 32℃, but reversibly change to hydrophilic below this temperature. According to the method of Rheinwald and Green, keratinocytes proliferated and made a multilayer on the grafted surfaces at 37℃, as on the nongrafted culture dishes. The multilayered keratinocyte sheets were detached from the grafted surfaces only by reducing temperature to 20℃ without need for dispase. No cell remnants were observed on the dishes. Such cell sheet detachment was not observed on nongrafted dishes. Immunoblotting of harvested keratinocyte sheets revealed that dispase treatment disrupted E-cadherin and laminin 5, while these molecules remained intact in the keratinocyte sheets harvested by only reducing temperature from the grafted dishes. Transmission electron microscopy revealed that desmosomes were destroyed in dispase treatment but retained in low-temperature treatment. Use of thermo-responsive dishes was examined as a new tool for tissue engineering to achieve the preparation of artificial epithelium for cell transplantation as well as for the investigation of intact multilayered keratinocyte sheets. After the transplantation experiments with atymic rats, we have just started the clinical application of tissue-engineered epidermal cell sheets.