Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (1): 174-178.doi: 10.3969/j.issn.1673-8225.2011.01.040

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In vitro amplification and biological characterization of rabbit corneal limbal epithelial stem cells

Mo Lian-jie1, Ye Yu-feng2, Ke Li-qin1, Ren Wang-fang1, Zhang Chun-fang1, Wu Lian-bao1, Zhang Fang-hua1, Liu Xiao-ling3   

  1. 1Clinical Medicine of Hangzhou Normal University, Hangzhou  310036, Zhejiang Province, China
    2Department of Ophthalmology, Hangzhou First People’s Hospital, Hangzhou  310006, Zhejiang Province, China
    3Medical Research Center of Hangzhou Normal University, Hangzhou  310036, Zhejiang Province, China
  • Received:2010-10-15 Revised:2010-11-25 Online:2011-01-01 Published:2011-01-01
  • Contact: Liu Xiao-ling, Associate professor, Medical Research Center of Hangzhou Normal University, Hangzhou 310036, Zhejiang Province, China liu_xaoling2004@yahoo.com.cn
  • About author:Mo Lian-jie, Clinical Medicine of Hangzhou Normal University, Hangzhou 310036, Zhejiang Province, China Ye Yu-feng, Doctor, Associate chief physician, Department of Ophthalmology, Hangzhou First People’s Hospital, Hangzhou 310006, Zhejiang Province, China Mo Lian-jie and Ye Yu-feng contributed equally to this paper.
  • Supported by:

    the Scientific and Technological Innovation Foundation for College Students of Zhejiang Province, No. ZX09030100; the Climbing Project of Hangzhou Normal University, No. YS01210010

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Abstract:

BACKGROUND: How to establish a stable in vitro culture system, including location of corneal limbal epithelial stem cells, in vitro sample harvest, in vitro culture, vector selection, as well as identification methods, play a key role in corneal limbal epithelial stem cells culture.
OBJECTIVE: To culture the isolated rabbit corneal limbal epithelial stem cells and to identify the biological properties of cultured cells.
METHODS: The primary rabbit cornel limbal epithelial stem cells were isolated and cultured with tissue inoculation using human amniotic membrane as vector. The growth features of cells were observed under an inverted microscope. The morphology of cells was observed by hematoxylin-eosin staining and a scanning electron microscope. Furthermore, the monoclonal antibody AE5 and P63 two-step immunohistochemical staining were used to identify limbal epithelial stem cell protein expression.
RESULTS AND CONCLUSION: The rabbit corneal limbal epithelial stem cells could be successfully cultured and maintained a relatively high value-added potential in vitro. Rabbit corneal limbal epithelial stem cells cultured on the amniotic membrane pull netted cellular layer. The AE5 monoclonal antibody positive rate of primary cultured cells was about 5% and P63 monoclonal antibody positive up to 90%. AE5-positive rate increased and P63-positive rate decreased with the increase in the number of subculture. The rabbit limbal epithelial stem cells can be successful culture and amplified on human amniotic membrane in vitro by limbal tissue culture method. The cultured cells maintain the characteristics of corneal epithelial cells. The rabbit corneal limbal epithelial stem cells can form grafts on the amniotic membrane.

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