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.

Sustained Ca2+ mobilizations: A quantitative approach to predict their importance in cell-cell communication and wound healing

“…One of the limitations of our study is that the epithelium is stratified except under injured conditions where at the wound margin it is a single cell. While there are several stratified cultures systems available, the cells are either cultured over feeder layers or cultured in the presence of medium containing serum [32]. Since the cells are cultured in a wounded environment, it is unclear what the addition of a physical wound would mean.…”

Fabrication of transplantable corneal epithelial and oral mucosal epithelial cell sheets using a novel temperature-responsive closed culture device

“…The closed‐culture devices, which we call cell cartridges, were prepared as described previously (Nakajima et al ., ). Briefly, the cell cartridge frame was made using an injection‐molding method with polycarbonate.…”

“…Rabbit limbal tissues were isolated from Japanese white house rabbit ocular globes (purchased from Japan Lamb Ltd) and were incubated with 200 U/ml dispase II (Sanko Junyaku, Tokyo, Japan) at 37°C for 1 h. The separated epithelial layer was treated with 0.25% trypsin–0.01 m m EDTA solution (Nacalai Tesque, Kyoto, Japan). Resuspended primary cells were cultured on the temperature‐responsive culture surfaces in the cell cartridges and temperature‐responsive culture inserts (six‐well; CellSeed, Tokyo, Japan) at a density of 2 × 10 4 or 4 × 10 4 cells/cm 2 in a keratinocyte culture medium (KCM) (Nakajima et al ., ). Human oral mucosal epithelial cells (passage 1; purchased from ScienCell Research Laboratories) were cultured on the temperature‐responsive culture surfaces in the same manner as the rabbit corneal epithelial cells.…”

“…To optimize the process of fabricating tissue‐engineered cell sheets, stabilize the quality of individual products and reduce production costs, an automated culture system needs to be developed (Kino‐oka and Taya, ). We previously developed a novel closed‐culture device that has two culture layers as well as a culture insert, for fabricating stratified epithelial cell sheets in an automated culture system (Nakajima et al ., ).…”

Fabricating retinal pigment epithelial cell sheets derived from human induced pluripotent stem cells in an automated closed culture system for regenerative medicine

“…However, there are still several risks associated with cultivation using open systems, such as contamination and uncontrolled changes during proliferation; each time the culture medium is replaced or cultured cells are harvested, the system must be opened and exposed to the environment. Therefore, we have attempted to develop a “closed” automated culture system in which cells are never directly exposed to the external environment during the procedure; using this system, we previously achieved the automated generation and culture of corneal epithelial cell sheets [16,17] and oral mucosal epithelial cell sheets (Nishimura A, Submitted) in accordance with established manual laboratory culture protocols (S1A Fig). Our closed system is comprised of several distinct features.…”