Ker-CT (ATCC® CRL-4048)

Organism: Homo sapiens, human  /  Cell Type: keratinocyte  /  Tissue: foreskin  /  Disease: normal

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Organism Homo sapiens, human
Tissue foreskin
Cell Type keratinocyte
Product Format frozen
Morphology epithelial
Culture Properties adherent
Biosafety Level 2  [Cells contain SV40 viral DNA sequences]
Disease normal
Age neonatal
Gender male
Applications

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

Karyotype Cytogenetic analysis was performed on G-banded metaphase cells from the human cell line Ker-CT and two abnormal male clones were detected. Clone 1 demonstrated trisomy 5 and trisomy 20. Clone 2 demonstrated trisomy 5 and four copies of chromosome 20.
Images
Derivation The Ker-CT cell line was immortalized by human telomerase and CDK4 using retroviral pBABE-puro hTERT and pSRalphaMSU expressing mouse CDK4.
Clinical Data male
neonatal
Antigen Expression Positive for p63 (TP63) and Cytokeratin 5 (KRT5)
Comments The Ker-CT cell line is positive for telomerase, failed to senesce, and was proliferating after more than 250 population doublings 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  
Complete Growth Medium KGM-Gold™ BulletKit™(Lonza 00192060)
Subculturing Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation solutions for culture vessels of other sizes.
  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.

Medium Renewal: Every 2-3 days. 

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.

Cryopreservation
90% FBS, 10% DMSO
Culture Conditions Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
STR Profile D5S818:  12
D13S317: 11, 13
D7S820: 10, 11
D16S539: 9, 11
vWA: 15, 16
Amelogenin:
TPOX: 8
CSF1PO: 7, 11
TH01: 6, 9
Name of Depositor J Shay
References

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

Basic Documentation
Other Documentation
FAQ's
  1. Ker-CT subculture
    ATCC® CRL-4048 ™ Ker-CT should be subcultured/split when the cells reach 80% to 90% confluence, usually 3-4 days.
    Date Updated: 3/27/2014
  2. Ker-CT cryopreservation
    Ker-CT (ATCC® CRL-4048 ™) should be frozen in 90% FBS:10% DMSO.  Store the cryopreserved cells in the vapor phase of liquid nitrogen.
    Date Updated: 3/27/2014
  3. Ker-CT differentiation
    It has been shown that regulatory mechanisms of differentiation remain intact in Ker-CT cells (ATCC® CRL-4048 ™)  (PubMed: 12545164).  When the calcium concentration is i...
    Date Updated: 3/27/2014
  4. Ker-CT seeding density
    The optimal seeding density for ATCC® CRL-4048 ™ Ker-CT is 4,000 to 8,000 cells/cm
    Date Updated: 3/27/2014
  5. Ker-CT culture medium
    The recommended growth media for ATCC® CRL-4048 ™ Ker-CT  is Lonza KGM-Gold bullet kit (catalog number 192060), which is comprised of 500 mL KBM-Gold™, phenol red-free basal ...
    Date Updated: 3/27/2014
  6. Thawing Ker-CT cells
    Cryopreserved Ker-CT cells (ATCC® CRL-4048 ™) should be quickly thawed at 37
    Date Updated: 3/27/2014
  7. Antibiotics in Ker-CT medium

    Antibiotics are not recommended as they can mask contamination.


    Date Updated: 3/27/2014
Restrictions

This material requires that the Addendum for Commercial and For-Profit Organizations or the Addendum for Noncommercial and Academic Organizations be signed and returned to ATCC before shipment. The price listed above is for noncommercial and academic organizations only. Commercial and for-profit organizations should call for pricing.

References

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