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

Remove ａnd discard culture medium.

Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.

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 within 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.

Add 6.0 to 8.0 mL of complete growth medium ａnd aspirate cells by pipetting gently.

To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube ａnd spin at approximately 125 x g for 5 to 10 minutes.

Discard supernatant ａnd resuspend cells in fresh serum-free growth medium. Add appropriate aliquots of cell suspension to new culture vessels.

Place culture vessels in incubators at 37°C.

Subcultivation Ratio: 1:3 to 1:4

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, 3th edition, published by Alan R. Liss, N.Y., 1994.

培养条件：Keratinocyte-Serum Free medium with 5 ng/ml human recombinant EGF (do not filter) and 0.05 mg/ml bovine pituitary extract (Invitrogen, formerly GIBCO-BRL, Cat. No. 17005-042) and supplemented with 0.005 mg/ml insulin and 500 ng/ml hydrocortisone.