title: Functional human corneal endothelial cell sheets harvested from temperature-responsive culture surfaces.
abstract: This study reports a new method for fabricating bioengineered human corneal endothelial cell sheets suitable for ocular surgery and repair. We have initially cultured human corneal endothelial cells on type IV collagen-coated dishes and, after several passages, expanded cells were then seeded onto novel temperature-responsive culture dishes. Four weeks after reaching confluence, these cultured endothelial cells were harvested as intact monolayer cell sheets by simple temperature reduction without enzymatic treatment. Scanning electron microscopy indicated that these cells were primarily hexagonal with numerous microvilli and cilia, similar to the native corneal endothelium. The Na+, K+-ATPase pump sites were located at the cell borders as in vivo. Moreover, cell densities and numbers of pump sites were identical to those of in vivo human corneal endothelium under optimized conditions. A 3H-ouabain binding analysis demonstrated a linear proportionality for cell pump density between confluent cell densities of 575 cells/mm2 and 3070 cells/mm2. We also confirmed Na+, K+-ATPase activity in the sheets in vitro. Xenograft transplantation results showed that the fabricated sheets retain their function of maintaining proper stromal hydration in vivo. We have established a regimen to culture and proliferate human corneal endothelial cells and fabricate endothelial sheets ex vivo morphologically and functionally similar to the native corneal endothelium. Our results support the value of harvested cell sheets for clinical applications in ocular reconstructive surgery in patients with ocular endothelial decompensation.
authors.affiliation: Department of Ophthalmology, Osaka University Medical School, Suita, Japan.
authors.forename: Taizo
authors.initials: T
authors.lastname: Sumide
authors.forename: Kohji
authors.initials: K
authors.lastname: Nishida
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamato
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ide
authors.forename: Yasutaka
authors.initials: Y
authors.lastname: Hayashida
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Watanabe
authors.forename: Joseph
authors.initials: J
authors.lastname: Yang
authors.forename: Chinatsu
authors.initials: C
authors.lastname: Kohno
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kikuchi
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Maeda
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Watanabe
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* / Q000201:enzymology; D006801:Humans; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013696:Temperature; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D000254:Sodium-Potassium-Exchanging ATPase