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1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.05% (w/v) Trypsin-0.53mM EDTA solution.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually with 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. (Or neutralize with medium containing 10% fetal bovine serum).
5. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
6. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels. Seed cells on flasks precoated with a mixture of 0.01 mg/ml fibronectin, 0.03 mg/mL bovine collagen type I and 0.01 mg/mL bovine serum albumin.
7. Incubate cultures at 37°C.

Kahn CR, Rhim J. Human corneal epithelial cell lines with extended lifespan. US Patent 5,585,265 dated Dec 17 1996

Walker TL, Kahn CR. Human corneal epithelial cell lines with extended lifespan. US Patent 5,672,498 dated Sep 30 1997