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C. Kim, L. A. Oliveira, T. V. Chirila, . Zainuddin, I. R. Schwab, M. I. Rosenblatt; Bombyx mori Silk Fibroin as a Biomatrix Substrate for ex vivo Expansion of Human and Rabbit Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1884.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate a silk fibroin biomatrix as a substrate to promote corneal epithelial cell adhesion, expansion, and differentiation ex vivo.
Human (eye bank) and rabbit (freshly enucleated) corneoscleral tissue were used to obtain corneal epithelial cell suspensions via double enzymatic digestion (dispase followed by trypsin). Circular silk discs were cut from silk sheets using a surgical trephine and placed into tissue culture plates. The epithelial cell suspensions were added to the silk containing plates, and the cells were then allowed to attach and proliferate. Cell adhesion, replication, and differentiation were analyzed by phase-contrast microscopy and scanning electron microscopy. Immunodetection of p63 and cytokeratin 3 expression was used to evaluate the epithelial lineage of these cells.
Viable corneal epithelial cultures were obtained from both human and rabbit corneoscleral tissue. Phase contrast microscopy revealed that at lower plating densities, cells initially formed in clusters, which after 7-10 days coalesced into confluent, partially multilayered sheets. Cross-sections of confluent epithelial cells attached to the silk biomatrix showed well-attached cells of normal morphology. These adherent epithelial cells were almost exclusively p63 and/or cytokeratin 3 positive, indicating that they were of an epithelial lineage of varying degrees of differentiation. Scanning electron micrographs confirmed the attachment of the epithelial cell processes to pores within the silk substrate. The surface of these epithelial cells was covered by fine microvilli similar to those seen on the corneal surface.
Bombyx mori silk fibroin film is a novel biomatrix for the cultivation of human and rabbit primary corneal epithelial cells. The transparency and strength of this biodegradable substrate may make it ideal for the reconstruction of an injured ocular surface utilizing ex vivo expanded epithelium.
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