Cultivation of corneal epithelial cell sheets on canine amniotic membrane.
OBJECTIVE
To develop and assess canine corneal epithelial cell sheets cultivated from limbal stem cells on amniotic membrane.


METHODS
Canine corneal limbal segments were obtained from six beagle dogs. Cryopreserved denuded amniotic membranes (obtained from Miniature Dachshund and Cavalier King Charles Spaniel breeds) from which the epithelial cells were removed were used as scaffolds. The limbal segments were cultured on these amniotic membranes with 3T3 feeder cells for 2 weeks. The harvested corneal epithelial cell sheets were stained with H&E for histologic analysis. The harvested sheets were analyzed immunohistochemically using a corneal epithelium-specific marker keratin 3(K3) and putative stem cell markers ABCG2, p63, and vimentin.


RESULTS
Cultivated cells from the corneal limbal tissues reached confluency in 7-8 days. The cultivated cells adhered to the denuded amniotic membrane and formed a sheet. The cultivated cell sheet was transparent and consisted of five to eight layers. K3 was observed in all layers and ABCG2, p63, and vimentin were notably present in the basal layer of the cultivated canine epithelium by immunofluorescence.


CONCLUSIONS
Canine corneal epithelial cells were successfully cultivated on the canine amniotic membrane. The cultivated epithelial sheets contained putative stem cells in the basal layer and had a stratified epithelium.
Laboratory of Veterinary Emergency Medicine, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Eunryel
E
Nam
Ayaka
A
Takahashi
Naoki
N
Fujita
Keiko
K
Tsuzuki
Ryohei
R
Nishimura
D016475:3T3 Cells; D000650:Amnion / Q000502:physiology*; D000818:Animals; D018929:Cell Culture Techniques / Q000662:veterinary; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology*; D019573:Epithelium, Corneal / Q000166:cytology*; D051379:Mice