Making of Sheet of Culture Corneal Epithelium and Keratocyte by Thermal-response Cell Culture Dish.

我校生物医学工程研究开发了热反应培养皿。该技术允许在没有胰蛋白酶的薄片上收集细胞培养物。用这种方法，我们复制了一个角膜上皮细胞和角质细胞层，角膜上皮细胞和角质细胞。目前以3T3细胞为喂料制备角膜上皮细胞片。然而，在临床应用中，均质细胞或血清培养更可取。我们比较了大白兔和人的再生角膜上皮层：1)角化细胞作为供体与标准3T3细胞的比较；2)分别在培养液中加入匀浆血清，与标准FBS进行比较；3)不同数量的生长因子。同样地，将白兔和人的再生角质细胞层分别加入培养液中，并与标准FBS进行比较。在光学显微镜和透射电子显微镜下观察制备的细胞片。将再生的角膜上皮细胞片和角质细胞片移植到白兔体内。该程序使我们能够收集类似于收集3T3细胞片的再生角化细胞片。虽然细胞层可以成功地复制，但上皮和其他角膜层在组织学上没有明显的分裂，就像在真角膜中看到的那样。在FBS和其他均质血清中均可进行血清繁殖。在培养液中添加不同数量的生长因子是否会产生差异尚不清楚。目前，再生上皮层和角质细胞层均可移植到白兔角膜上。然而，再生的角质细胞层是否能替代原有物质尚不清楚。

The biomedical engineering research at our University has developed the thermal-response culture dish. The technique allows the collection of cell culture on a sheet without trypsins. With this method, we reproduced a corneal epithelium and a keratocyte layer with corneal epithelial cells and keratocyte cells. The sheets of corneal epithelial cells have so far been made with 3T3 cells as the feeder. However, in clinical applications, cultures from homogenous cells or serum are more preferable. We compared the reproduced corneal epithelium layers of white rabbits and of humans using : 1)keratocytes as the feeders as compared with the standard 3T3 cells ; 2)respective homogenous serum added to culture solutions as compared with the standard FBS ; and 3)different amount of growth factors. Similarly, reproduced keratocyte layers of white rabbits and humans using respective homogenous serum added to culture solutions as compared with the standard FBS were examined. The cell sheets made were examined under an optical microscope and a transmission electron microscope. The reproduced corneal epithelial cell sheets and the keratocyte sheets were transplanted to a white rabbit. The procedure allowed us to collect a reproduced keratocyte sheet similar to the collection of a 3T3 cell sheet. Although a cell layer could be reproduced successfully, a clear histological division of epithelium and other corneal layers as seen in a real cornea was not found. Serum reproduction was possible in both FBS and other homogenous serums. Whether a different amount of growth factor added to the culture solution could make a difference was unclear. At present, a reproduced epithelium layer and a keratocyte layer could be transplanted to the cornea of a white rabbit. However whether a reproduced keratocyte layer can substitute the original substance is unclear.