title: 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.
  
  
  METHODS
  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.
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Eunryel
authors.initials: E
authors.lastname: Nam
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Fujita
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Maresuke
authors.initials: M
authors.lastname: Morita
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Tsuzuki
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Hsing Yi
authors.initials: HY
authors.lastname: Lin
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Cheng Shu
authors.initials: CS
authors.lastname: Chung
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nakagawa
authors.affiliation: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Nishimura
mesh_terms: D016475:3T3 Cells; D000650:Amnion / Q000378:metabolism*; D000818:Animals; D016667:Bacterial Capsules / Q000378:metabolism; D002478:Cells, Cultured / Q000166:cytology; D003094:Collagen / Q000378:metabolism*; D004285:Dogs / Q000033:anatomy & histology*; D019573:Epithelium, Corneal / Q000166:cytology*; D005455:Fluorescent Antibody Technique / Q000662:veterinary; D005782:Gels; D019394:Ki-67 Antigen / Q000378:metabolism; D051379:Mice; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D013234:Stem Cells / Q000166:cytology*
chemical_list: D005782:Gels; D019394:Ki-67 Antigen; C059821:atelocollagen; C098418:capsular polysaccharide K3; D003094:Collagen
keywords: amniotic membrane; collagen gel; corneal epithelium; dog; limbus; temperature-responsive culture dish