Removal of viable sheets of conjunctival epithelium with dispase II.

A technique (based on Gipson and Grill, Invest Ophthalmol Vis Sci 23:269, 1982) has been developed to obtain pure, viable, intact sheets of rabbit conjunctival epithelium free of the underlying basement membrane. After preparing a full thickness eye wall resection, Dispase, grade II (neutral protease-Bacillus polymyxa), 1.2 Units/ml MEM is injected intrasclerally. The conjunctiva and sclera are pinned in agar and incubated in the dispase with MEM for 1 hour. A 2 X 3 mm sheet of conjunctival epithelium can be dissected bluntly. Light microscopy shows a two- to three-layered epithelium with many goblet cells. Transmission electron microscopy reveals blebbing at the freed basal epithelial cell membrane, intact desmosomes, and intact goblet cells. The conjunctival sheets were cultured on epithelial-scraped corneal stromal carriers in vitro. Numerous goblet cells were present up to 12 hours on 4-mm carriers and 24 hours on 1-mm carriers. With this technique, pure populations of conjunctival epithelium can be isolated for further characterization and tissue culture.