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Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

Note: The flasks used should be 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. Subculture when 80% confluent.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.05% Trypsin – EDTA (GIBCO cat# 25300-054).
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  fresh medium and aspirate cells by gently pipetting. 
5. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to10 minutes.
6. Discard supernatant and resuspend cells in fresh growth medium.  Add appropriate aliquots of cell suspension to new coated culture vessels. 
7. Place culture vessels in incubators at 37°C.

Subcultivation Ratio: 1:3
Medium Renewal: Every 2 to 3 days.

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.  

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

Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.