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Clonal growth of these cells is improved by using a 3T3  (ATCC CCL-92) feeder layer (see Rheinwald and Green, Cell 6:331, 1975 for methods) RefRheinwald JG, Green H. Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma. Cell 6: 317-330, 1975. PubMed: 1052770.

ATCC grows these cells on 56-X.2, MITC-STO cells. The feeder cells must be plated 24 hours before use at 2 X 10⁶/T75 in order to obtain a 30% confluent monolayer.

• 400 ng/ml hydrocortisone
• fetal bovine serum to a final concentration of 10%.

Note: Subculture before 100% confluent.

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

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
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 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.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. 
5. Add appropriate aliquots of the cell suspension to new culture vessels pre-plated with ATCC 56-X.2™ feeder layer (MITC-STO cells).
6. Inoculate new flasks with 3 x 10³ cells/cm².
7. Incubate cultures at 37°C.

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Animal Cells: A Manual Of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.  

Medium Renewal
Two to three times weekly

Rheinwald JG, Beckett MA. Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultures from human squamous cell carcinomas. Cancer Res. 41: 1657-1663, 1981. PubMed: 7214336

Rheinwald JG, Beckett MA. Defective terminal differentiation in culture as a consistent and selectable character of malignant human keratinocytes. Cell 22: 629-632, 1980. PubMed: 6160916

Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

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

Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.