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

TLDR

Cell sheet-based tissue engineering requires automation that optimizes fabrication, ensures quality, and minimizes contamination, which a closed culture system can achieve. The study develops a closed culture device, a cell cartridge, for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on porous membranes with 3T3 feeder cells in the cartridge, and five membrane thicknesses were tested to produce stratified sheets, which were then histologically characterized. Stratified corneal epithelial sheets were successfully produced using a 25 µm gas‑permeable membrane, with appropriate marker expression, indicating the cartridge’s effectiveness. © 2012 John Wiley & Sons, Ltd.

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. Copyright © 2012 John Wiley & Sons, Ltd.