A new method to obtain epithelial and stromal explants from human Corneo-Scleral Discs for the routine culture of corneal epithelial and fibroblast cells.

Acquisition of human corneal cells for culture is hindered not only by the scarcity of donor tissues but also by some of the standard enzymatic and mechanical isolation techniques. Good yields have been reported from full-thickness explant and sclero-limbal pieces. However, due to their greater proliferative capacity, fibroblasts will encroach and subsequently overwhelm epithelial cultures whichever technique is used. The novel approach presented here is to minimise this by removal of the whole stroma from the epithelial layers at the outset. This is achieved by selective sectioning with the Webb mini-microtome developed in the Norwich Eye Research Laboratory. The microtome can be sterilised by alcohol spraying or autoclaving and is small enough to use in the culture hood. A selective cut in the region of the Bowman's membrane results in the isolation of the epithelium from the stroma and thus exposed, the basal epithelial layers are released from contact inhibition to allow growth. The stroma is further cut to produce multiple sections for the culture of fibroblasts. Both pure epithelial and stromal fibroblast cultures have been successfully generated in serum-enriched medium as well as defined serum-free media with growth supplements, from the corneo-scleral discs of donors of all ages.