April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Differentiation Status of Bovine Limbal Epithelial Cells Cultured on Intact and Denuded Amniotic Membrane Before and After Air-Lifting
Author Affiliations & Notes
  • B. Chen
    School of Pharmacy, University of Reading, Reading, United Kingdom
  • S. Mi
    School of Pharmacy, University of Reading, Reading, United Kingdom
  • B. Wright
    School of Pharmacy, University of Reading, Reading, United Kingdom
  • C. J. Connon
    School of Pharmacy, University of Reading, Reading, United Kingdom
  • Footnotes
    Commercial Relationships  B. Chen, None; S. Mi, None; B. Wright, None; C.J. Connon, None.
  • Footnotes
    Support  University of Reading RETF, Cardiff University (School of Optometry and Vision Science), BBSRC (BB/F019742/1)
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 358. doi:
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      B. Chen, S. Mi, B. Wright, C. J. Connon; Differentiation Status of Bovine Limbal Epithelial Cells Cultured on Intact and Denuded Amniotic Membrane Before and After Air-Lifting. Invest. Ophthalmol. Vis. Sci. 2010;51(13):358.

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Abstract

Purpose: : To investigate differences in limbal epithelial cell differentiation when expanded upon intact (amniotic epithelial cells remaining) and denuded human amniotic membrane, a commonly used substrate in ophthalmic surgery for corneal stem cell transplantation.

Methods: : Animal and human tissue were handled in accordance with the statement for the use of animals in ophthalmic and visual research and declaration ofHelsinki. Ex vivo expansion of the limbal epithelial cells, in supplemented media (50µg/ml antibiotics, 5% FBS, 0.5% dimethyl sulfoxide, 2ng/ml human Epidermal Growth Factor, 5µg/ml insulin, B27 supplement medium) at 37°C, 5% CO2, continued for 2 weeks followed by 1 week under ‘air-lifting’ condition. Before and after air-lifting the differentiated (K3/K12 positive) and undifferentiated (K14 positive) cells were examined by immunohistochemistry, Western blotting and real time PCR.

Results: : Limbal epithelial cells expanded upon amniotic membrane formed 4-6 stratified layers, both on intact and denuded amniotic membrane (n>6). On denuded amniotic membrane the proportion of differentiated cells remained unaltered following air-lifting, i.e. the level of K3 protein and K12 mRNA were similar. However, within cells grown on intact amniotic membrane the number of differentiated cells increased significantly following air-lifting (>200 fold in K12 mRNA level) in marked contrast cells grown on denuded amniotic membrane

Conclusions: : AM preparation can markedly influence a measured degree of differentiation within limbal epithelia cells following airlifting.

Keywords: cornea: epithelium • differentiation • extracellular matrix 
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