Production of clinically applicable tissue-engineered cornea

为了生产出临床适用的组织工程化角膜，需要从角膜的三层中每一层获取组织干细胞。首先，我们证明了通过神经球实验可以从角膜的三层中的每一层获得组织干细胞。然后，我们研究了这些组织干细胞在动物模型中的应用方法。我们将来自人角膜内皮的微球注入兔的前房和附着在兔角膜后表面的微球，以保持角膜的透明度。目的：探讨以40μm胶原膜作为Descemet膜替代载体进行人角膜内皮细胞片状移植的可行性。这些结果为胶原膜用于角膜内皮失代偿的培养的人脐静脉内皮细胞移植的可行性提供了证据。我们将人角膜基质中的球体接种到40-50μm的多孔明胶薄膜上，并将其移植到兔的角膜基质袋中。然后，人角膜基质微球产生了丰富的细胞外基质，如层粘连蛋白和纤维连接蛋白，这表明人角膜基质微球在临床上是可行的。我们比较了兔角膜内皮细胞前体在角膜中央和周边区域的分布和自我更新能力。我们的发现表明，外周和中央兔角膜内皮细胞都含有大量的前体，但相对于中央区，外周内皮细胞含有更多的前体细胞，具有更强的自我更新能力。

To produce clinically applicable tissue-engineered cornea, it is required to obtain tissue stem cells from each of three layers of the cornea. First, we showed that tissue stem cells can be obtained from each of three layers of the cornea by neuro-sphere assay. Then, we investigated the method of applying these tissue stem cells in animal models. We injected spheres from human corneal endothelium into the anterior chamber of rabbits and the spheres attached to the back surface of rabbit cornea, maintaining the transparency of the cornea. To evaluate the feasibility of a method of human corneal endothelial cells (HCEC) sheet transplantation using collagen membrane of 40 μm as a substitute carrier of Descemet's membrane. These results provide evidence for the feasibility of cultured HCEC sheet transplantation using collagen membrane for corneal endothelial decompensation. We seeded spheres from human corneal stroma onto the porous gelatin sheets of 40-50μm and transplanted the sheet into the corneal stromal pockets of rabbits. Then, the spheres from human corneal stroma produced abundant of extracellular matrix such as laminin and fibronectin, indicative of the clinical feasibility of spheres from human corneal stroma. We compared the distribution and self-renewal capacity of rabbit corneal endothelial cell precursors in the central and peripheral regions of the cornea. Our findings demonstrate that both peripheral and central rabbit corneal endothelium contain a significant number of precursors, but the peripheral endothelium contains more precursors with a stronger self-renewal capacity relative to the central region.