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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
  1. Yoshinori Oie1,2,
  2. Ryuhei Hayashi1,
  3. Ryo Takagi3,
  4. Masayuki Yamato3,
  5. Hiroshi Takayanagi4,
  6. Yasuo Tano2,
  7. Kohji Nishida1
  1. 1Department of Ophthalmology and Visual Science, Tohoku University Graduate School of Medicine, Sendai, Japan
  2. 2Department of Ophthalmology, Osaka University Medical School, Suita, Japan
  3. 3Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan
  4. 4Translational Research Center, Tohoku University, Sendai, Japan
  1. Correspondence to Dr Kohji Nishida, Department of Ophthalmology and Visual Science, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan; knishida{at}oph.med.tohoku.ac.jp)

Abstract

Background/aims 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.

Conclusion This novel culture method using dermal fibroblasts and pharmaceutical agents provides a safe cell processing system without xenogenic feeder cells for ocular surface reconstruction.

  • Clinically approved supplements
  • cornea
  • culture method
  • dermal fibroblast
  • experimental and laboratory
  • ocular surface
  • oral mucosal epithelial cells
  • stem cells

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Footnotes

  • Funding This study was funded by the Ministry of Health Labor and Welfare, and the Ministry of Education, Culture, Sports, Science and Technology in Japan.

  • Competing interests None.

  • Ethics approval This study was conducted with the approval of the institutional review board of Tohoku University School of Medicine.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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