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E. O. Graue, B. D. Lawrence, D. L. Kaplan, I. R. Schwab, M. I. Rosenblatt; Expansion of Corneal Epithelial Cells on a Silk Fibroin Biomatrix. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5752. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the ability of silk fibroin biomaterials to support the expansion of corneal limbal explants and corneal epithelial cell suspensions.
Limbal tissue was isolated from freshly sacrificed New Zealand white rabbits. For limbal explant experiments, 2 X 2 mm limbal sections were incubated for 40 minutes in Dispase II (1.2 UI/ml) and the tissue samples placed on silk discs, denunded amniotic membrane segments or on the plastic surface of culture dishes. For cell suspension experiments, limbal sheets were further treated with trypsin-EDTA and rendered into a single cell suspension via trituration. Cell proliferation was compared between explants grown on silk fibroin sheets and those grown on plastic by serial imaging of cultures every day for 2 weeks. For proliferation and cell adhesion assays, 6 mm diameter silk films were coated with either collagen (10 µg/cm2), laminin (0.16 mg/ml), or poly-D-lysine (10 µg/ml) for 24 hours at 4oC. Rabbit corneal epithelial cells were plated on pre-coated films at a density of 1x103 cells/cm2. Cell adhesion to silk fibroin films at day 1 was measured by counting the number of cells per field of view after gentle washing three times with rabbit corneal epithelial cell media . Cell proliferation was measured at 72 hours using the MTT cell proliferation assay.
Limbal explants attached and expanded on both silk films and plastic tissue culture dishes and reached confluency at similar rates. Cells cultured on denuded amniotic membrane achieved confluency at a slower rate than those expanded on either silk films or plastic surfaces. The mean number of cells/field were similar on collagen (38.5±2.71), laminin (34.5±2.8), and uncoated (25.5±2.9) films, while significantly more cells adhered to poly-D-lysine (76±7.4) coated silk. Cells on the poly-D-lysine substrate did not proliferate, however. Using the MTT assay, the proliferation of epithelial cells on plastic, uncoated silk, or silk coated with collagen or laminin was not statistically different.
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