title: Comparison of cell-suspension and explant culture of rabbit limbal epithelial cells.
abstract: Currently, most investigators directly use limbal explants to culture corneal epithelial cells. However, it has not been identified that limbal stem cells do readily migrate from the limbal explants onto culture plate or amniotic membrane carrier. In this study a cell-suspension culture system for rabbit limbal stem cells was developed and compared with the direct explant method in the aspect of stem cells content in the culture system. Rabbit limbal epithelial cells were dissociated from rabbit eyes by dispase and single cell suspension was made for cell-suspension culture. DeltaNp63 expression of cultured rabbit limbal epithelial cells by cell-suspension technique and explant technique was detected. In cell-suspension culture, isolated cell-suspension was evaluated by flow cytometric analysis for vimentin expression and residual limbal tissue after dispase treatment was examined by scanning electron microscopy. In limbal epithelial cells suspension less than 5% cells were vimentin positive. Examination of residual limbal tissue confirmed that all the limbal epithelial cells had been removed. Histological examination revealed that with cell-suspension culture the cultured epithelial cells could differentiate better than with explant technique. In cells cultured with cell-suspension, there were much more cells expressing DeltaNp63 than in explant cultured cells. In cells cultured with explants, most of DeltaNp63 labelling cells mustered around the explants, and peripheral cells on the slides were DeltaNp63 negative. These results suggested that with pure limbal epithelial cells suspension including basal cells, which could directly enter into culture system, cell-suspension culture technique was significantly superior to explant culture technique in terms of stem cells content.
authors.affiliation: Eye Centre of Tianjin Medical College, 64#, Tongan road, Heping district, Tianjin 300070, People's Republic of China.
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Zhang
authors.forename: Huimin
authors.initials: H
authors.lastname: Sun
authors.forename: Xin
authors.initials: X
authors.lastname: Tang
authors.forename: Jian
authors.initials: J
authors.lastname: Ji
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Li
authors.forename: Jing
authors.initials: J
authors.lastname: Sun
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Ma
authors.forename: Jiaqin
authors.initials: J
authors.lastname: Yuan
authors.forename: Zhong Chao
authors.initials: ZC
authors.lastname: Han
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D019573:Epithelium, Corneal / Q000378:metabolism / Q000648:ultrastructure*; D016850:Limbus Corneae / Q000378:metabolism / Q000648:ultrastructure*; D008855:Microscopy, Electron, Scanning; D010750:Phosphoproteins / Q000378:metabolism; D011817:Rabbits; D013234:Stem Cells / Q000378:metabolism / Q000648:ultrastructure; D015534:Trans-Activators / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
chemical_list: D010750:Phosphoproteins; D015534:Trans-Activators; C486419:Trp63 protein, mouse; D014746:Vimentin