Surgical grafts and methods of preparation
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United States of America Patent
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Issued Date -
Feb 3, 2005
app pub date -
Apr 28, 2004
filing date -
Apr 28, 2003
priority date (Note) -
Abandoned
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Abstract
The present invention relates to the discovery of a method of enzymatically, consistently and reproducibly, isolating a viable, intact limbal epithelial sheet that retains stem cell characteristics in the basal epithelium. The method comprises the steps of: a) obtaining limbus from a biopsy of a donor eye of a living individual, a living-related individual, or from a cadaveric eye, the limbus comprising limbal epithelium and an underlying stroma; b) contacting the limbus with a solution comprising Dispase 2, for a period of time and under conditions sufficient to loosen a limbal epithelial sheet from the stroma, thereby forming a loosely adherent limbal epithelial sheet; and c) mechanically separating the loose epithelial sheet from the underlying stroma, thereby isolating a substantially intact, viable, limbal epithelial sheet. Also disclosed are a new culture system to achieve ex vivo expansion of human corneal keratocytes while maintaining their characteristic dendritic morphology and continuous expression of keratocan even in the presence of high concentrations of serum by growing them on the stromal matrix of the human amniotic membrane (AM), and a surgical graft comprising keratocytes on amniotic membrane.
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