March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
The Potential Of Non-animal Derived Culture Components In Ex Vivo Expansion Of Human Limbal Epithelial Cells
Author Affiliations & Notes
  • Amanda J. Vernon
    Institute of Ophthalmology, University College London, London, United Kingdom
  • Stefan Schrader
    Institute of Ophthalmology, University College London, London, United Kingdom
  • Julie T. Daniels
    Institute of Ophthalmology, University College London, London, United Kingdom
  • Footnotes
    Commercial Relationships  Amanda J. Vernon, None; Stefan Schrader, None; Julie T. Daniels, None
  • Footnotes
    Support  National Institute for Health Research Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital and UCL Institute of Ophthalmology
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1819. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Amanda J. Vernon, Stefan Schrader, Julie T. Daniels; The Potential Of Non-animal Derived Culture Components In Ex Vivo Expansion Of Human Limbal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1819.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose: : Cultured limbal epithelial stem cell (LESC) therapy has been used to treat blinding ocular surface disease associated with LESC failure. Graft production methods are not optimal, removing animal-derived products from the culture system would improve graft safety. This study investigates limbal epithelial cell (LEC) expansion in media, supplemented with human serum (HS) as an alternative to animal-derived foetal calf serum (FCS). In addition LECs were cultured with human MRC5 foetal lung fibroblasts in order to compare their efficacy to gold-standard mouse 3T3 fibroblasts.

Methods: : LECs were isolated from allogeneic cornea-scleral rims using enzymatic digestion and mechanical scraping. LECs were cultured with MMC growth arrested 3T3 feeder cells with the addition of corneal epithelial culture media (CECM), supplemented with either 10% HS or FCS. LEC characteristics were examined by light microscopy and colony forming efficiency (CFE) assay. The expression of putative LESC and differentiation markers (p63α and CK3) were determined using immunocytochemistry. Further LEC cultures were cultured in the presence of MMC growth arrested human MRC5 or mouse 3T3 fibroblasts. LEC characteristics were examined by light microscopy and CFE.

Results: : LECs cultured with HS produced large, round, smooth colonies indicative of colonies formed by poorly differentiated cells. CFE indicated HS maintained a population of LECs with proliferative capacity. Light microscopy showed small cells with typical epithelial-like cobblestone morphology exhibiting a large nuclear to cytoplasmic (N/C) ratio. HS-cultured LECs expressed both p63α and CK3. LECs expanded on human MRC5 fibroblasts maintained a population of LECs with proliferative capacity. Light microscopy showed small cells with typical epithelial-like cobblestone morphology exhibiting a large N/C ratio.

Conclusions: : LECs maintained a poorly differentiated phenotype and proliferative capacity when cultured in HS, or when expanded on human MRC5 fibroblasts. Further validation is required to use these components within a clinical setting to ensure these components are not only safe but also effective; therefore this study is a step forward in improving such cell therapy products to patients.

Keywords: cornea: epithelium • transplantation • regeneration 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×