title: Structural characterization of bioengineered human corneal endothelial cell sheets fabricated on temperature-responsive culture dishes.
abstract: For the purpose of corneal regenerative medicine, we fabricated human corneal endothelial cell sheets on temperature-responsive dishes, which could be non-invasively harvested as intact, transplantable sheets by simply reducing the culture temperature. Cells demonstrated hexagonal cell shape with numerous microvilli and cilia, and also exhibited abundant cytoplasmic organelles similar to these cells in vivo. Immunofluorescence for type IV collagen and fibronectin revealed that abundant extracellular matrix (ECM) was deposited on the basal surface throughout culture, and the deposited ECM was harvested along with the cell sheets by reducing culture temperature to 20 degrees C. Faint ECM remnants were observed on the dish surfaces after cell sheet detachment. Immunofluorescence for ZO-1 showed that tight junctions were established between cells, and immunoblotting indicated that intact ZO-1 was maintained during cell sheet harvest, while conventional proteolytic cell harvest methods resulted in the degradation of ZO-1. These results suggest that these transplantable corneal endothelial cell sheets can be applied to treat patients with damaged corneas.
authors.affiliation: Department of Ophthalmology, Osaka University Medical School, Osaka, 2-2 Yamadaoka, Rm. E7, Suita, Osaka 565-0871, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ide
authors.forename: Kohji
authors.initials: K
authors.lastname: Nishida
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamato
authors.forename: Taizo
authors.initials: T
authors.lastname: Sumide
authors.forename: Mika
authors.initials: M
authors.lastname: Utsumi
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nozaki
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kikuchi
authors.forename: Teruo
authors.initials: T
authors.lastname: Okano
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Tano
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000166:cytology* / Q000502:physiology*; D005109:Extracellular Matrix / Q000502:physiology* / Q000648:ultrastructure*; D006801:Humans; D013696:Temperature; D023822:Tissue Engineering / Q000379:methods*