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

角膜内皮细胞对保持视力非常重要。但是，它们在出生后的增长有限。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，角膜发生水肿。导致角膜混浊和视力下降，需要角膜移植。日本的角膜供体不足，我们需要获得更多的角膜。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型细胞培养表面，该表面会随着温度的变化而改变其表面疏水性。虽然该表面与商业聚苯乙烯细胞培养表面具有相似的疏水性，并且在37℃时有利于细胞的粘附和增殖，但接枝聚合物在32℃以下变得亲水，并且释放出不含胰蛋白酶的扩散培养细胞。将SD大鼠和Balb/c小鼠的角膜内皮细胞在37℃的温度下在这些表面培养至融合。当培养温度降低到32℃以下时，细胞从pipaam嫁接区域分离，不需要胰蛋白酶。在没有使用蛋白酶的情况下，仅仅通过降低温度就完整地收获了这片角膜。薄片基底面的细胞-细胞连接和细胞外基质，对薄片的完整性和功能至关重要，保持完整。我们尝试使用没有FBS的培养基。采用穿透性角膜移植术（同种异体内皮和自体间质）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).