Development of Transplantable Corneal Epithelium Fabricated with Cell Sheet Engineering

角膜缘干细胞缺乏症是由眼外伤或疾病引起的，可导致角膜混浊和视力丧失。最近已经尝试制造角膜上皮移植物结构，但该技术仍在发展中。我们已经开发了一种新的细胞片操作技术，使用温度响应培养表面产生功能，培养角膜上皮细胞片移植。将人或兔角膜缘干细胞与丝裂霉素C处理的3 T3饲养层在37℃的温度响应培养皿中共培养。2周后，通过将温度降至20℃，从培养皿中收获细胞片。进行组织学分析、免疫印迹和集落形成测定以表征细胞片。采用自体角膜缘干细胞移植重建实验性角膜缘干细胞缺乏的兔角膜表面。简单地通过降低温度而不使用蛋白酶来完整地收获多层角膜上皮片。细胞-细胞连接和细胞外基质的基础侧的表，至关重要的表的完整性和功能，保持完整。在薄片中发现了存活的角膜祖细胞群，其数量接近最初接种的数量。收获的薄片易于操作，无需任何载体即可移植，并且易于粘附于角膜基质，因此不需要角膜基质。兔角膜表面重建非常成功。细胞片工程技术使我们能够创造完整的，可移植的角膜上皮细胞片，保留干细胞从角膜缘干细胞体外扩增。我们的研究表明，我们的生物工程角膜上皮片具有非常有前途的临床能力。

Limbal stem-cell deficiency by ocular trauma or diseases causes corneal opacification and visual loss. Recent attempts have been made to fabricate corneal epithelial graft constructs, but the technology is still evolving. We have developed a novel cell-sheet manipulation technology using temperature-responsive culture surfaces to generate functional, cultivated corneal epithelial cell sheet grafts. Human or rabbit limbal stem cells were cocultured with mitomycin C-treated 3T3 feeder layers on temperature-responsive culture dishes at 37℃. Cell sheets were harvested from the dishes after 2 weeks by reducing temperature to 20℃. Histologic analyzes, immunoblotting, and colony-forming assay were performed to characterize the cell sheets. Autologous transplantation was undertaken to reconstruct the corneal surfaces of rabbits with experimentally induced limbal stem cell deficiencies. Multilayered corneal epithelial sheets were 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. A viable population of corneal progenitor cells, close in number to that originally seeded, was found in the sheets. Harvested sheets were easily manipulated, transplantable without any carriers, and readily adhesive to corneal stroma so that suturing was not required. Corneal surface reconstruction in rabbits was highly successful. Cell sheet engineering technology allows us to create intact, transplantable corneal epithelial cell sheets that retain stem cells from limbal stem cells expanded ex vivo. Our research indicates highly promising clinical capabilities for our bioengineered corneal epithelial sheet.