After 55 population doublings, the cells continue dividing and retain cuboidal morphology. E6E7 sequences were detected by PCR in cells at passage 15.

Major Histocompatibility Complex class I or II molecules were not expressed on these cells, but PCR analyses revealed presence of the genes for directing synthesis of HLA antigens.

1. xa0Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mMxa0 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).xa0Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.xa0 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.xa0
5. Add appropriate aliquots of the cell supension to new culture vessels.
6. Incubate cultures at 37°C.

Medium Renewal:xa0Two to three times weekly.

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. xa0xa0

Zabrenetzky V, et al. The isolation, immortalization and characterization of human fetal keratinocytes. In Vitro Cell. Dev. Biol. 33: Part II, p. 34A, 1997.