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所研制了热响应培养皿。这项技术允许在没有胰酶的情况下在一张纸上收集细胞培养物。用这种方法，我们复制了一个角膜上皮和一个含有角膜上皮细胞和角质细胞的角质细胞层。到目前为止，角膜上皮细胞片是以3T3细胞作为饲养层制成的。然而，在临床应用中，用同种细胞或血清培养更可取。我们比较了白兔和人的再生角膜上皮层：1)以角膜基质细胞为饲养层，与标准3T3细胞相比较；2)在培养液中分别加入匀浆，与标准胎牛血清相比较；以及3)不同数量的生长因子。类似地，用加入到培养液中的各自的均质血清与标准的FBS相比，检测了白兔和人的再生角膜细胞层。制备的细胞片在光学显微镜和透射电子显微镜下进行了观察。将再生的角膜上皮细胞片和角膜上皮细胞片移植到大白兔体内。该程序允许我们收集复制的角质细胞片，类似于收集3T3细胞片。尽管细胞层可以成功复制，但没有发现像在真实角膜中看到的那样，上皮和其他角膜层在组织学上的明显分裂。血清复制在FBS和其他均一血清中都是可能的。目前尚不清楚在培养液中加入不同数量的生长因子是否会产生不同的效果。目前，可以将再生上皮层和角质细胞层移植到白兔的角膜上。然而，复制的角质细胞层是否可以替代原始物质尚不清楚。

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.