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.