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

为了开发无需酶处理即可获取可移植培养上皮的新技术，将人角质形成细胞接种在接枝了热响应性聚合物聚N-异丙基丙烯酰胺的培养皿中。接枝后的碟子表面在32℃以上略有疏水，但在此温度以下可逆地变为亲水。根据Rheinwald和Green的方法，在37℃时，角质形成细胞在移植的表面上增殖并形成多层，与未移植的培养皿上的一样。只需将温度降至20℃，即可将多层角质形成细胞片从移植表面上分离出来，而不需要进行任何处理。培养皿上未见细胞残留物。在未移植的培养皿中未观察到这种细胞膜的脱落。对收获的角质形成细胞片的免疫印迹显示，消解酶处理破坏了E-钙粘附素和层粘连蛋白5，而这些分子在仅从嫁接的培养皿中降温获得的角质形成细胞片中保持完整。透射电子显微镜显示，酶处理后桥粒被破坏，低温处理时桥粒仍被保留。利用热敏培养皿作为一种新的组织工程工具来实现细胞移植所需的人工上皮的制备以及完整的多层角质形成细胞片的研究。经过同种异体大鼠的移植实验，组织工程化表皮细胞片的临床应用才刚刚开始。

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.