title: A novel closed cell culture device for fabrication of corneal epithelial cell sheets.
abstract: Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.
authors.affiliation: Central Research Laboratory, Hitachi Ltd, 2520 Hatoyama, Saitama, 350-0395, Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Nakajima
authors.affiliation: Central Research Laboratory, Hitachi Ltd, 2520 Hatoyama, Saitama, 350-0395, Japan.
authors.forename: Toyoshige
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Central Research Laboratory, Hitachi Ltd, 2520 Hatoyama, Saitama, 350-0395, Japan.
authors.forename: Noboru
authors.initials: N
authors.lastname: Moriya
authors.affiliation: CellSeed Inc, 33-8 Wakamatsu-cho, Shinjuku-ku, Tokyo, 162-0056, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Mizutani
authors.affiliation: Central Research Laboratory, Hitachi Ltd, 2520 Hatoyama, Saitama, 350-0395, Japan.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Kan
authors.affiliation: Central Research Laboratory, Hitachi Ltd, 2520 Hatoyama, Saitama, 350-0395, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nozaki
authors.affiliation: Central Research Laboratory, Hitachi Ltd, 2520 Hatoyama, Saitama, 350-0395, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Saitoh
authors.affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamato
authors.affiliation: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan.
authors.forename: Teruo
authors.initials: T
authors.lastname: Okano
authors.affiliation: Central Research Laboratory, Hitachi Ltd, 2520 Hatoyama, Saitama, 350-0395, Japan.
authors.forename: Shizu
authors.initials: S
authors.lastname: Takeda
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001331:Automation; D018929:Cell Culture Techniques / Q000295:instrumentation*; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D062445:Claudin-1 / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology*; D004867:Equipment Design; D061252:Feeder Cells; D005740:Gases; D007150:Immunohistochemistry; D053558:Keratin-3 / Q000378:metabolism; D051379:Mice; D008858:Microscopy, Phase-Contrast; D010100:Oxygen / Q000737:chemistry; D010539:Permeability; D010750:Phosphoproteins / Q000378:metabolism; D016062:Porosity; D011817:Rabbits; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D062445:Claudin-1; D005740:Gases; D053558:Keratin-3; D010750:Phosphoproteins; D010100:Oxygen