Production of clinically applicable tissue-engineered cornea

为了生产临床上可应用的组织工程角膜，需要从角膜的三层中的每一层获得组织干细胞。首先，我们证明了组织干细胞可以通过神经球分析从角膜的三层中的每一层获得。然后，我们研究了将这些组织干细胞应用于动物模型的方法。我们将人角膜内皮细胞的微球注入兔前房，微球附着在兔角膜的后表面，保持角膜的透明性。目的探讨以40 μm胶原膜作为角膜后弹力层替代载体的人角膜内皮细胞片移植方法的可行性。这些结果为胶原膜移植培养的内皮细胞片治疗角膜内皮失代偿的可行性提供了证据。我们将人角膜基质球接种于40-50μm的多孔明胶片上，并将其移植到兔角膜基质袋中。然后，来自人角膜基质的球体产生了丰富的细胞外基质，例如层粘连蛋白和纤连蛋白，这表明来自人角膜基质的球体的临床可行性。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。我们的研究结果表明，外周和中央兔角膜内皮细胞含有大量的前体，但外周内皮细胞含有更多的前体，具有更强的自我更新能力相对于中央区域。

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.