June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Development of the cell sheet production technology from cryopreserved human corneal epithelial cells; the quality test and in vivo study
Author Affiliations & Notes
  • Kiwamu Imagawa
    Research Division, JCR Pharmaceuticals, Kobe, Japan
  • Kenichi Maeda
    Research Division, JCR Pharmaceuticals, Kobe, Japan
  • Shuichi Yokoyama
    Research Division, JCR Pharmaceuticals, Kobe, Japan
  • Yuki Hosoda
    Research Division, JCR Pharmaceuticals, Kobe, Japan
  • Shunsuke Watanabe
    Research Division, JCR Pharmaceuticals, Kobe, Japan
  • Takahiro Nakamura
    Research Center for Inflammation and Regenerative Medicine Faculty of Life and Medical Sciences, Doshisha University, Kyoto, Japan
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Shigeru Kinoshita
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Footnotes
    Commercial Relationships Kiwamu Imagawa, None; Kenichi Maeda, None; Shuichi Yokoyama, None; Yuki Hosoda, None; Shunsuke Watanabe, None; Takahiro Nakamura, None; Shigeru Kinoshita, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 5635. doi:
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      Kiwamu Imagawa, Kenichi Maeda, Shuichi Yokoyama, Yuki Hosoda, Shunsuke Watanabe, Takahiro Nakamura, Shigeru Kinoshita; Development of the cell sheet production technology from cryopreserved human corneal epithelial cells; the quality test and in vivo study. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5635.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The purpose of this study is to develop cell sheet manufacturing technology using cryopreserved human corneal epithelial cells. We evaluate the quality of the cell sheets in vitro and confirm the suitability of the cell sheets in vivo in limbal stem cell deficiency rabbit model.

Methods: Primary corneal epithelial cells were cultured in serum-free medium with supplements. After detaching by enzymatic treatment, cells were collected and frozen. One of the vials was thawed for quality assessment. Once cells were confirmed to meet the criteria for quality, cells were thawed and cultured to produce cell sheets. The cell sheets were transplanted onto denuded rabbit corneas and monitored for 2 weeks. The engrafted cell sheets were recovered and analyzed by immunohistochemistry.

Results: We found freezing and thawing does not affect the viability and cell growth of corneal epithelial cells expanded in primary culture. By seeding the post-thawed cells on denuded amniotic membrane, we were able to produce cell sheets with stratified structure. The transplanted cell sheets remained transparent, smooth, and without epithelial defects during the observation period of two weeks.

Conclusions: Based on the results of in vitro quality tests and in vivo study, the cell sheets using post-thawed cells were confirmed to have the same quality as the cell sheets produced by conventional method. By expanding corneal epithelial cells in primary culture, the number of cell sheets manufactured from one cornea dramatically increased. Moreover, by using cryopreserved cells that meet the certain criteria for quality, it is considered to reduce variation in the quality of the cell sheets.The transplantation of cultivated cell sheets produced with cryopreserved cells could be a promising technique for the treatment of limbal failure.

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