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Ker-CT

CRL-4048

Product category
Human cells
Product type
hTERT-immortalized cell
Organism
Homo sapiens, human
Cell type
keratinocyte
Morphology
epithelial
Tissue
Skin; Foreskin
Disease
Normal
Applications
3D cell culture
Drug development
High-throughput screening
Toxicology
Product format
Frozen
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Documentation

Biosafety Icon BSL 2

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

Cells contain SV40 sequences

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Detailed product information

General

Specific 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

Characteristics

Growth properties
Adherent
Derivation
The Ker-CT cell line was immortalized by human telomerase and CDK4 using retroviral pBABE-puro hTERT and pSRalphaMSU expressing mouse CDK4.
Age
neonate
Gender
Male
Immortalization method
hTERT expression and mouse cyclin dependent kinase 4 (CDK4) expressing retrovirus
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.
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  

Handling information

Unpacking and storage instructions
  1. Check all containers for leakage or breakage.
  2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.
Complete medium
KGM-Gold™ BulletKit™(Lonza 00192060)
Temperature
37°C
Atmosphere
95% Air, 5% CO2
Handling procedure
To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt.
If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor phase and NOT
at –70°C. Storage at –70°C will result in loss of viability.
  1. Prepare a 25 cm2 or a 75 cm2 culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents, the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
  2. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
  3. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All operations from this point on should be carried out under strict aseptic conditions.
  4. Thaw the frozen vial at 37°C and resuspend the cells in 5mL of complete growth medium.  Count the cells and seed at recommended seeding densities. DO NOT centrifuge after thawing to remove DMSO. 
  5. Incubate the culture at 37°C in a suitable incubator.
  6. Change to fresh medium after the cells attached, usually 6-12 hours later, to remove DMSO and FBS.
Subculturing procedure
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.

Reagents for cryopreservation
Fetal bovine serum supplemented with 10% (v/v) DMSO (ATCC 4-X)

Quality control specifications

STR profiling
Amelogenin: X,Y
CSF1PO: 7,11
D13S317: 11,13
D16S539: 9,11
D5S818: 12
D7S820: 10,11
TH01: 6,9
TPOX: 8
vWA: 15,16

History

Depositors
J Shay

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

Material Transfer Agreement Addendum for Screening Applications

For-profit organizations
For every order of this item, you must provide a signed Material Transfer Agreement Addendum for Screening Applications. We cannot ship this item until we receive this addendum. The person signing the addendum as the principal investigator must match the end user as listed on the applicable sales order for the item.

Email the signed addendum to [email protected] with a reference to both your account and sales order numbers. Once received, your addendum will be reviewed, and this item will be released for shipment if all requirements are met. Additional fees may apply if this product is being used for a screening use (ATCC ACS-2103F), and these fees will be applied after your order is confirmed. If you need assistance with your order, please contact our Customer Care team or your applicable distributor.

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Curated Citations

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

Frequently Asked Questions

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