A novel closed cell culture device for fabrication of corneal epithelial cell sheets

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. Copyright © 2012 John Wiley & Sons, Ltd.

[1]  Masakazu Yamamoto,et al.  Prevention of esophageal stricture after endoscopic submucosal dissection using tissue-engineered cell sheets. , 2012, Gastroenterology.

[2]  Masakazu Yamamoto,et al.  Fabrication and Validation of Autologous Human Oral Mucosal Epithelial Cell Sheets to Prevent Stenosis after Esophageal Endoscopic Submucosal Dissection , 2011, Pathobiology.

[3]  Y. Tabata,et al.  A novel gelatin hydrogel carrier sheet for corneal endothelial transplantation. , 2011, Tissue engineering. Part A.

[4]  H. Onoe,et al.  Fabrication of transplantable 3D-neuronal network , 2010 .

[5]  Masayuki Yamato,et al.  Validation system of tissue-engineered epithelial cell sheets for corneal regenerative medicine. , 2010, Tissue engineering. Part C, Methods.

[6]  M. Yamato,et al.  A novel method of culturing human oral mucosal epithelial cell sheet using post-mitotic human dermal fibroblast feeder cells and modified keratinocyte culture medium for ocular surface reconstruction , 2010, British Journal of Ophthalmology.

[7]  Hiroyuki Honda,et al.  A compact, automated cell culture system for clinical scale cell expansion from primary tissues. , 2010, Tissue engineering. Part C, Methods.

[8]  Masahito Taya,et al.  Recent developments in processing systems for cell and tissue cultures toward therapeutic application. , 2009, Journal of bioscience and bioengineering.

[9]  T. Okano,et al.  Transportation of transplantable cell sheets fabricated with temperature‐responsive culture surfaces for regenerative medicine , 2008, Journal of tissue engineering and regenerative medicine.

[10]  Ashok Kumar,et al.  Skin tissue engineering for tissue repair and regeneration. , 2008, Tissue engineering. Part B, Reviews.

[11]  Masayuki Yamato,et al.  Reconstruction of functional tissues with cell sheet engineering. , 2007, Biomaterials.

[12]  K. Tsubota,et al.  Hypoxia enhances the expansion of human limbal epithelial progenitor cells in vitro. , 2007, Investigative ophthalmology & visual science.

[13]  R. Rice,et al.  Oxygen-dependent differentiation of human keratinocytes. , 2007, The Journal of investigative dermatology.

[14]  R. Soetikno,et al.  Treatment of oesophageal ulcerations using endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in a canine model , 2006, Gut.

[15]  Hideo Namiki,et al.  The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets. , 2006, Biomaterials.

[16]  S. Mizuno,et al.  Hydrostatic pressure/perfusion culture system designed and validated for engineering tissue. , 2005, Journal of bioscience and bioengineering.

[17]  Masayuki Yamato,et al.  Ocular surface reconstruction using autologous rabbit oral mucosal epithelial sheets fabricated ex vivo on a temperature-responsive culture surface. , 2005, Investigative ophthalmology & visual science.

[18]  T. Okano,et al.  Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. , 2004, The New England journal of medicine.

[19]  N. Fullwood,et al.  Tight junction-related protein expression and distribution in human corneal epithelium. , 2003, Experimental eye research.

[20]  G. Pellegrini,et al.  Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency. , 2001, Transplantation.

[21]  D. Ponzin,et al.  p63 identifies keratinocyte stem cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[22]  R. Tsai,et al.  Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. , 2000, The New England journal of medicine.

[23]  Michele De Luca,et al.  Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium , 1997, The Lancet.

[24]  A. Schermer,et al.  Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells , 1986, The Journal of cell biology.

[25]  宮下 英之.  A novel NIH/3T3 duplex feeder system to engineer corneal epithelial sheets with enhanced cytokeratin 15-positive progenitor populations , 2009 .

[26]  T. Okano,et al.  Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells , 2006, Journal of Artificial Organs.

[27]  大木 岳志,et al.  Treatment of oesophageal ulcerations using endoscopic transplantation of tissue-engineered autologous oral mucosal epithelial cell sheets in a canine model , 2006 .