Ocular surface reconstruction using autologous rabbit oral mucosal epithelial sheets fabricated ex vivo on a temperature-responsive culture surface.
OBJECTIVE
Autologous stem cell transplantation for total limbal stem cell deficiency is immunologically preferable, to avoid allograft rejection. This study was undertaken to investigate the possibility of a novel tissue engineering approach for ocular surface reconstruction, using autologous oral mucosal epithelial stem cells expanded ex vivo on temperature-responsive cell culture surfaces.


METHODS
Rabbit oral mucosal epithelial cells cultured on temperature-responsive culture surfaces with mitomycin-C-treated 3T3 feeder cells for 2 weeks produced confluent epithelial cell sheets. Putative progenitor cell populations were estimated by colony-forming assays. Autologous transplantation of these cell sheets to surgically manipulated eyes was performed, and ocular surface reconstruction and cell phenotypic modulation were examined.


RESULTS
All cultured oral epithelial cells were nonenzymatically harvested as transplantable intact cell sheets by reducing culture temperature to 20 degrees C. Oral epithelial cells were stratified in three to five cell layers more similar to corneal epithelium than to oral mucosal epithelium. Colony-forming assays and immunofluorescence for p63, beta1-integrin, and connexin 43 indicated retention of viable stem and/or progenitor cell populations in cell sheets. Autologous transplantation to rabbit corneal surfaces successfully reconstructed the corneal surface, with restoration of transparency. Four weeks after transplantation, epithelial stratification was similar to that in the corneal epithelium, although the keratin expression profile retained characteristics of the oral mucosal epithelium.


CONCLUSIONS
Cell sheet harvest technology enables fabrication of viable, transplantable, tissue-engineered epithelial cell sheets that retain putative progenitor cells from autologous oral mucosal epithelial cells. Promising clinical capabilities for autologous tissue-engineered epithelial cell sheets for ocular surface reconstruction are indicated.
Hayashida
Yasutaka
Y
Department of Ophthalmology, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Nishida
Kohji
K
Yamato
Masayuki
M
Watanabe
Katsuhiko
K
Maeda
Naoyuki
N
Watanabe
Hitoshi
H
Kikuchi
Akihiko
A
Okano
Teruo
T
Tano
Yasuo
Y
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D017690:Cell Transplantation / Q000379:methods*; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D003316:Corneal Diseases / Q000473:pathology / Q000601:surgery*; D004847:Epithelial Cells / Q000637:transplantation*; D019084:Fluorescent Antibody Technique, Indirect; D006085:Graft Survival; D007633:Keratins / Q000378:metabolism; D009061:Mouth Mucosa / Q000166:cytology*; D011817:Rabbits; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology; D023822:Tissue Engineering; D014182:Transplantation, Autologous
D015415:Biomarkers; D007633:Keratins