title: Carrier-free epithelial cell sheets prepared by enzymatic degradation of collagen gel.
abstract: Limbal stem-cell deficiency by ocular trauma or disease causes corneal opacification and vision loss. Conventional tissue engineering using biodegradable scaffolds has met with limited success. In this study, we developed a novel method for preparing carrier-free epithelial cell sheets, which have potential for use in repairing defects of the ocular surface. Stratified corneal epithelial cell sheets were prepared in culture dishes coated with biodegradable type I collagen. Haematoxylin and eosin staining, electron microscopy and immunohistochemistry were performed to characterize the cell sheets. Then, carrier-free epithelial sheets were successfully engineered using specific collagenase to degrade the collagen gel. Cell sheets of four to six cell layers after culture for 14 days were similar to natural rabbit corneal epithelia, as shown by pathological examination. Microvillus, tight cell-cell junctions and desmosome junctions were observed via electron microscopy. K3 and basement membrane components, such as type IV collagen and laminin, were expressed in the cells sheets and integrin β1 was maintained in basal cells. This novel method of using collagenase to degrade collagen gel is both simple and effective in preparing intact carrier-free epithelial cell sheets. Such sheets have great potential for application during in vivo corneal regeneration.
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre, Sun Yat-Sen University, Guangzhou, People's Republic of China.
authors.forename: Qicheng
authors.initials: Q
authors.lastname: Ke
authors.forename: Xiaoran
authors.initials: X
authors.lastname: Wang
authors.forename: Qianying
authors.initials: Q
authors.lastname: Gao
authors.forename: Zheng
authors.initials: Z
authors.lastname: Wu
authors.forename: Pengxia
authors.initials: P
authors.lastname: Wan
authors.forename: Weijiao
authors.initials: W
authors.lastname: Zhan
authors.forename: Jian
authors.initials: J
authors.lastname: Ge
authors.forename: Zhichong
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism*; D024141:Collagen Type IV / Q000378:metabolism; D003315:Cornea / Q000166:cytology / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000201:enzymology*; D005131:Eye Injuries / Q000378:metabolism / Q000628:therapy*; D005782:Gels; D005786:Gene Expression Regulation; D007797:Laminin / Q000096:biosynthesis; D011817:Rabbits; D012038:Regeneration; D023822:Tissue Engineering
chemical_list: D024042:Collagen Type I; D024141:Collagen Type IV; D005782:Gels; D007797:Laminin