CP-C (CP-94251) (ATCC® CRL-4029)

Organism: Homo sapiens, human  /  Cell Type: Epithelial cells immortalized with hTERT  /  Tissue: Esophagus; epithelium  /  Disease: Cancer, Barrett's esophagus

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Organism Homo sapiens, human
Tissue Esophagus; epithelium
Cell Type Epithelial cells immortalized with hTERT
Product Format frozen
Morphology Epithelial-like
Culture Properties Adherent
Biosafety Level 2
Disease Cancer, Barrett's esophagus
Age Adult
Gender Male
Applications
This well-characterized pre-malignant culture represents a unique tool for studying esophageal cancer progression.
Storage Conditions Liquid nitrogen vapor phase
Karyotype Terminal restriction fragment lengths (TRF) analyses show the cells have increased telomerase activity and extended telomeres of about 12 kb. Morphologically, the cell line is similar to early passage cultures exhibiting smaller cells with large nucleus to cytoplasm ratio. Genetic instability studies using flow cytometry and FISH reveal the retention of elevated tetraploidy (G2/tetraploidy) in the hTERT-immortalized cells similar to the non-transduced parental cells [PubMed: 12807723]. RefPalanca-Wessels MC, et al. Extended lifespan of Barrett's esophagus epithelium transduced with the human telomerase catalytic subunit: a useful in vitro model. Carcinogenesis 24(7): 1183-1190, 2003. PubMed: 12807723
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Derivation
The Barrett's esophagus cell line, CP-C (also identified as CP-94251) was derived from an endoscopic biopsy specimen obtained from a region of high-grade dysplasia. The cells were immortalized by transduction with a retroviral expression vector, pLXSN-hTERT, to create an immortalized cell line.
Antigen Expression

Antigen expression: This cell line is positive for epithelial marker pan-cytokeratin and negative for gastric mucin (CLH2) as assessed by immunocytochemistry (verified at ATCC).

Complete Growth Medium The base medium for this cell line is MCDB-153. To make the complete growth medium, add the following components to the base medium:
  • 0.4 µg/ml hydrocortisone
  • 20 ng/ml recombinant human EGF (Epidermal Growth Factor)
  • 8.4 µg/L cholera toxin
  • 20 mg/L adenine
  • 140 µg/ml BPE (Bovine Pituitary Extract)
  • 1x ITS Supplement (Sigma; I1884) [5 µg/ml Insulin; 5 µg /ml Transferrin; 5 ng/ml Sodium Selenite; final concs.]
  • 4 mM glutamine
  • fetal bovine serum to a final concentration of 5%

Note: To prepare Cholera toxin (Stock 100 µg/mL) : 0.5 mg Cholera toxin (Sigma C8052) + 5 mL dH20.  Aliquot into microcentrifuge tubes.   Add 84 µL of this 100 µg/mL stock solution to 1L of MCDB-153 base medium.

Subculturing
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. Add 2.0 to 3.0 mL of 0.25% (w/v) Trypsin - 0.53 mM 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.
  3. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  4. Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes. Discard supernatant.
  5. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 2 X 104 to 3 X 104 viable cells/cm2 is recommended.
  6. Incubate cultures at 37°C. Subculture when cells reach a concentration between 7 X 104 and 1 X 105 cells/cm2.
Subcultivation ratio: A subcultivation ratio of 1:2 to 1:4 is recommended.
Medium renewal: every 3 to 4 days

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Aminal Cells: A Manual of Basic Techniques by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.
Cryopreservation
Freeze medium: RPMI-1640 Medium, 80%; fetal bovine serum, 10%; DMSO, 10%
Storage temperature: Liquid nitrogen vapor phase
Culture Conditions
Temperature: 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
STR Profile
CSF1PO: 9, 13
D13S317: 8, 10
D16S539: 11, 12
D5S818: 12
D7S820: 8, 11
THO1: 8, 9.3
TPOX: 8
vWA: 17
Amelogenin: XY
Population Doubling Level (PDL) As part of our quality control, we have tested this cell line for its ability to grow for a minimum of 15 population doublings after recovery from cryopreservation. We have also compared its karyotype, telomerase expression level, growth rate, morphology and tissue-specific markers when first recovered from cryopreservation with that of cells at 10+ population doublings to ensure that there is no change in these parameters and that the cells are capable of extended proliferation.
Name of Depositor B Reid
Year of Origin September 1995
References

Palanca-Wessels MC, et al. Genetic Analysis of Long-term Barrett's Esophagus Epithelial Cultures Exhibiting Cytogenetic and Ploidy Abnormalities. Gastroentrology 114:114-295, 1998. PubMed: 9453489

Barrett MT, et al. Molecular Phenotype of Spontaneously Arising 4N (G2-Tetraploid) Intermediates of Neoplastic Progression in Barrett's Esophagus. Cancer Res. 63: 4211-4217, 2003. PubMed: 12874028

Maley CC, et al. Genetic clonal diversity predicts progression to esophageal adenocarcinoma. Nat. Genet. 38(4): 468-473, 2006. PubMed: 16565718

Palanca-Wessels MC, et al. Extended lifespan of Barrett's esophagus epithelium transduced with the human telomerase catalytic subunit: a useful in vitro model. Carcinogenesis 24(7): 1183-1190, 2003. PubMed: 12807723

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
  • License agreement required for commercial customer uses.
  • This material is distributed for research purposes only. A signed addendum to the ATCC Material Transfer Agreement must be sent to ATCC in advance of shipment.
Basic Documentation
Other Documentation
Restrictions

This material is subject to claims under U.S. Patent Nos. 6,261,836 and 6,337,200, other pending patent applications, and foreign counterparts thereof. It is required that either the Addendum for Commercial and For-Profit Organizations or the Addendum for Noncommercial and Academic Organizations, as appropriate, be signed and returned to ATCC before shipment.

References

Palanca-Wessels MC, et al. Genetic Analysis of Long-term Barrett's Esophagus Epithelial Cultures Exhibiting Cytogenetic and Ploidy Abnormalities. Gastroentrology 114:114-295, 1998. PubMed: 9453489

Barrett MT, et al. Molecular Phenotype of Spontaneously Arising 4N (G2-Tetraploid) Intermediates of Neoplastic Progression in Barrett's Esophagus. Cancer Res. 63: 4211-4217, 2003. PubMed: 12874028

Maley CC, et al. Genetic clonal diversity predicts progression to esophageal adenocarcinoma. Nat. Genet. 38(4): 468-473, 2006. PubMed: 16565718

Palanca-Wessels MC, et al. Extended lifespan of Barrett's esophagus epithelium transduced with the human telomerase catalytic subunit: a useful in vitro model. Carcinogenesis 24(7): 1183-1190, 2003. PubMed: 12807723