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Overexpression of CDK4 and hTERT in neonatal foreskin keratinocyte induced a dramatic upregulation of p16INK4a and milder upregualtion of p53 and p21WAF1, which became unresponsive to UV irradiation.  Despite the high levels of these checkpoint factors, Ker-CT cells divide in an apparently normal regulated fashion, are able to respond to changes in calcium levels, retain the stem cell phenotype, and fully differentiate and stratify in organotypic culture RefRamirez R, et al. Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells. Oncogene 22(3): 433-44, 2003. PubMed: 12545164

1. Remove and discard spent medium.
2. Briefly rinse with HBSS (ATCC 30-2213), 1 mL/25 cm² and discard rinse solution.
3. Add trypsin for primary cells (ATCC PCS-999-003), 1mL / 25cm².  Place at 37oC for 4-6 minutes, until 90% of the cells have detached.  
4. Rap flask gently to ensure cells are detached. Add 2% FBS in D-PBS, 1 mL/25cm² to neutralize trypsin. 
5. Centrifuge cells at 250 x g for 5 min at room temperature.
6. Remove supernatant. Resuspend pellet in 6.0 to 8.0 mL Complete Growth Medium.
7. Count cells, and seed 5.0 x 10³ to 8.0 x 10³ viable cells/cm² to new culture vessels.

As the cells become more confluent, increase the volume of media as follows: under 25% confluence feed cells 5 mL per 25 cm², 25-45% confluence then feed cells 7.5 mL per 25 cm², over 45% confluence then feed cells 10 mL per 25 cm².

Ramirez R, et al. Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells. Oncogene 22(3): 433-44, 2003. PubMed: 12545164

Vaughan M, et al. H-ras expression in immortalized keratinocytes produces an invasive epithelium in cultured skin equivalents. PLoS One 4(11): e7908, 2009. PubMed: 19936293