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

从不含蛋白水解酶的热响应细胞培养皿中非侵入性收获新型可移植表皮细胞片

基本信息

批准号：
13557148
负责人：
OKANO Teruo
金额：
$ 8.06万
依托单位：
Tokyo Women's Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

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.

为了开发用于在没有分散酶处理的情况下收获可移植的培养上皮的新技术，将人角质形成细胞铺在用温敏聚合物聚（N-异丙基丙烯酰胺）接枝的培养皿上。在32℃以上，接枝后的培养皿表面呈微疏水性，但在32 ℃以下，接枝后的培养皿表面可逆地变为亲水性。根据Rheinwald和绿色的方法，在37℃下，角质形成细胞在移植表面上增殖并形成多层膜，与在未移植的培养皿上一样。仅通过将温度降低至20℃而不需要分散酶来将多层角质形成细胞片层从移植表面分离。在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片脱离。收获的角质形成细胞片的免疫印迹显示分散酶处理破坏了E-钙粘蛋白和层粘连蛋白5，而这些分子在仅通过降低温度从移植皿收获的角质形成细胞片中保持完整。透射电子显微镜观察发现，桥粒在分散酶处理中被破坏，但在低温处理中保留。使用热响应皿作为一种新的工具，组织工程，以实现人工上皮细胞移植的准备，以及完整的多层角质形成细胞片的调查进行了检查。在无胸腺大鼠移植实验之后，组织工程表皮细胞片的临床应用才刚刚开始。