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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.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the corneal epithelial cell growth rate and phenotype on collagen vitrigels into which growth factors and peptides have been incorporated.
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.
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.
This study supports previous results that both plain and unmodified vitrigels may be useful for in vitro reconstruction of the corneal epithelium.
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