A technique for obtaining sheets of intact rabbit corneal epithelium.

We have developed a technique for obtaining sheets of intact epithelium from rabbit corneas. Nine-millimeter corneal buttons are removed and placed in culture medium containing 1.2 U/ml Dispase II, a bacterial neutral protease. The posterior half of the stroma is removed with forceps. The anterior half is incubated in the Dispase medium for 1 hr at 35 degrees C. The epithelial sheet is then removed by gentle probing with forceps between the epithelium and the stroma. Sheets so obtained have intact basal cells and desmosomes, and the free cell surface of basal cells send out cytoplasmic blebs. The action of the enzyme appears to be at the level of hemidesmosome basement membrane attachment. Polarity of the sheets is easily determined because the cut edges of the sheet curl inward toward the apical surface. These sheets provide excellent viable epithelium for studies of epithelial adhesion and synthesis and pure epithelium for culture.