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.
OBJECTIVE
To cultivate human oral mucosal epithelial cell sheets with post-mitotic human dermal fibroblast feeder cells and modified keratinocyte culture medium for ocular surface reconstruction.


METHODS
Human oral mucosal epithelial cells obtained from three healthy volunteers were cultured with x-ray-treated dermal fibroblasts (fibroblast group) and NIH/3T3 feeder layers (3T3 group) on temperature-responsive culture dishes. Media were supplemented using clinically approved products. Colony-forming efficiency was determined in both groups. Histological and immunohistochemical analyses were performed for cell sheets. Cell viability and purity of cell sheets were evaluated by flow cytometry.


RESULTS
Colony-forming efficiency in the fibroblast group was similar to that in the 3T3 group. All cell sheets were well stratified and harvested successfully. The expression patterns of keratin 1, 3/76, 4, 10, 12, 13, 15, ZO-1 and MUC16 were equivalent in both groups. The percentage of p63-positive cells in the fibroblast group (46.1+/-4.2%) was significantly higher than that in the 3T3 group (30.7+/-7.6%) (p=0.038, t test). The cell viability and purity were similar between the two groups.


CONCLUSIONS
This novel culture method using dermal fibroblasts and pharmaceutical agents provides a safe cell processing system without xenogenic feeder cells for ocular surface reconstruction.
Department of Ophthalmology and Visual Science, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan.
Yoshinori
Y
Oie
Ryuhei
R
Hayashi
Ryo
R
Takagi
Masayuki
M
Yamato
Hiroshi
H
Takayanagi
Yasuo
Y
Tano
Kohji
K
Nishida
D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology; D019573:Epithelium, Corneal / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D009061:Mouth Mucosa / Q000166:cytology / Q000637:transplantation*
D015415:Biomarkers