Comparison of the canine corneal epithelial cell sheets cultivated from limbal stem cells on canine amniotic membrane, atelocollagen gel, and temperature-responsive culture dish.

Abstract

OBJECTIVE: The current study compared canine corneal epithelial cell sheets cultivated from limbal stem cells on amniotic membrane, atelocollagen gel, and temperature-responsive culture dish. PROCEDURES: We collected limbal epithelial cells from the intact eyes of beagles and cultivated the cells on denuded canine amniotic membranes, temperature-responsive cell culture labware, and collagen gel with 3T3 feeder cells. Immunofluorescence staining for Ki-67 was used to analyze the capacity of cell proliferation in the sheets. Immunofluorescence staining was also performed for the corneal epithelium-specific marker cytokeratin 3 and putative stem cell markers ABCG2 and p63. Reverse-transcription polymerase chain reaction (RT-PCR) was performed to detect ABCG2 and p63. RESULTS: The growth rates of the cultivated cells, or the times it took them to reach confluency, were different for the three scaffolds. The cultivated sheet on the temperature-responsive dish consisted of 2-3 layers, while those on the collagen gel and on the amniotic membrane consisted of 5-8 layers. The basal layer cells grown on all three scaffolds expressed putative stem cell markers. In real-time RT-PCR analysis, the highest level of p63 was observed in the sheets grown on collagen gel. CONCLUSIONS: In this study, the cells cultured on the collagen gel demonstrated a capacity for cell proliferation, and the expressions of stem cells in the sheets suggested that collagen gel is the most suitable carrier for clinical use.