THE CORNEAL ENDOTHELIAL CELL CULTURE AND ENDOTHELIAL TRANSPLANTATION USING TEMPERATURE-RESPONSIVE CULTURE DISHES

角膜内皮细胞对清晰的视力非常重要。但是，出生后增加有限。当角膜营养不良、炎症和/或损伤导致角膜内皮细胞数量减少时，角膜就会水肿。导致角膜混浊和视力下降，需要进行角膜移植。东京女子医科大学生物医学工程研究所此前通过接枝聚（N-异丙基丙烯酰胺）（PIPAAm）开发了一种温度响应性细胞培养表面，该表面可响应温度改变其表面疏水性。虽然该表面显示出与商业聚苯乙烯细胞培养表面相似的疏水性，并且在37 ℃下有利于细胞粘附和增殖，但接枝聚合物在32 ℃以下变得亲水，并且释放分散的培养细胞而不含胰蛋白酶。当培养温度降低到32 ℃以下时，细胞从PIPAAm移植区域脱离，而不需要任何胰蛋白酶。该角膜片仅通过降低温度而完整地收获，而不使用蛋白酶。细胞-细胞连接和细胞外基质的基础侧的表，至关重要的表的完整性和功能，保持完整。我们尝试使用不含FBS的培养基。使用穿透性角膜移植术技术（同种异体内皮和自体基质）对小鼠进行角膜移植。

The corneal endothelial cells are very important for clear vision. But, they have limited to increase after birth. When the numbers of corneal endothelial cells decrease by corneal dystrophies, inflammation and/or injury, the cornea became edema. That causes corneal opacification and visual loss and need the corneal transplantation. The donor corneas are insufficient in Japan and we need to get more corneas.Institute of Biomedical Engineering, Tokyo Women's Medical University have previously developed a temperature-responsive cell culture surface by grafting poly(N-isopropylacrylamide)(PIPAAm) that changes its surface hydrophobicity in response to temperature. While this surface shows similar hydrophobicity to that of commercial polystyrene cell culture surfaces and facilitates cell adhesion and proliferation at 37 degrees C, grafted polymer becomes hydrophilic below 32 degrees C and releases spread cultured cells without trypsin.The corneal endothelial cells from SD rat or Balb/c mouse were cultured to confluency at 37 degrees C on these surfaces. When the culture temperature was reduced below 32 degrees C, cells detached from the PIPAAm-grafted areas without any need for trypsin. This Corneal sheet was harvested intact simply by reducing the temperature, without the use of proteases. Cell-cell junctions and extracellular matrix on the basal side of the sheet, critical to sheet integrity and function, remained intact. We tried to use the medium without FBS. Corneal transplantation performed for mouse using the Penetrating Keratoplasty technique (allogenic endothelium and autogenic stroma).