May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Corneal Epithelial Cell Growth Over Protein/Peptide Bulk-Modified Collagen Vitrigels
Author Affiliations & Notes
  • W. McIntosh Ambrose
    Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland
  • J. Wang
    Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland
  • S. Ng
    Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland
  • T. Takezawa
    Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Tsukuba, Japan
  • J. Elisseeff
    Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland
  • Footnotes
    Commercial Relationships  W. McIntosh Ambrose, None; J. Wang, None; S. Ng, None; T. Takezawa, Inventor of Vitrigel Technology, P; J. Elisseeff, None.
  • Footnotes
    Support  Coulter Foundation, NSF Graduate Research Fellowship, UNCF-Merck Graduate Research Fellowship
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 5750. doi:https://doi.org/
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      W. McIntosh Ambrose, J. Wang, S. Ng, T. Takezawa, J. Elisseeff; Corneal Epithelial Cell Growth Over Protein/Peptide Bulk-Modified Collagen Vitrigels. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5750. doi: https://doi.org/.

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

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Abstract

Purpose: : To evaluate the corneal epithelial cell growth rate and phenotype on collagen vitrigels into which growth factors and peptides have been incorporated.

Methods: : Modified collagen vitrigels were made by adding the following growth factors and peptides prior to the gelation step of vitrigel fabrication: epidermal growth factor (EGF) at 0.5, 2.5 and 5 µg/ml, nerve growth factor (NGF) at 0.05, 0.1 and 0.2 µg/ml, YIGSR at 2.6x10-2, 1.0x10-1 µg/ml, RGD at 50, 100, 200 µg/ml, PHSRN at 0.75, 1.5, 3.0 µg/ml and FAPP at 50, 100, 200 µg/ml. Primary rabbit corneal epithelial cells were cultured on the surface of both modified and control vitrigels, as well as tissue culture plastic. Cell growth was evaluated over a 5 to 7 day culture period, after which the cells were immunostained with antibodies against epithelial cytokeratin AE5 and zona occludens-1 (ZO-1). F-actin filaments were visualized using phalloidin.

Results: : Transparency of the 20-50µm thick vitrigels was maintained despite the modification procedures. Epithelial cells on all culture surfaces reached confluence by the end of the culture period, however, the growth rate for control and modified vitrigels was significantly higher than that of TCP cultures. All cultures stained positively for AE5, and showed focal adhesions as demonstrated by F-actin staining. ZO-1 staining was localized to both the cell membrane and nucleus, with some cells on NGF modified surfaces showing increased membrane localized staining in regions corresponding to F-actin banding.

Conclusions: : This study supports previous results that both plain and unmodified vitrigels may be useful for in vitro reconstruction of the corneal epithelium.

Keywords: cornea: epithelium • growth factors/growth factor receptors • microscopy: light/fluorescence/immunohistochemistry 
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