CP-D (CP-18821) (ATCC® CRL-4030™)

Organism: Homo sapiens, human  /  Tissue: Esophagus; Epithelium  /  Cell Type: high-grade dysplasia

Organism Homo sapiens, human
Tissue
Esophagus; Epithelium
Cell Type high-grade dysplasia
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 This is a hypotetraploid human cell line with the following derivative chromosomes consistently present at several different passages: add(2)(q13), der(3)t(3;8)(p10;q10), ider(7)(q10)dup(q31), der(12)t(12;13)(p10;q10), der(14)t(14;15)(q10;q10), der(15)t(15;22)(q10;q10), add(22)(q13)x2. In addition, there were consistent losses of one copy of chromosomes X, 10, 13, 14, 15, 19 and 20. Other less consistent structural aberrations were observed in some of the examined cells.
Images
Derivation
The Barrett's esophagus cell line, CP-D (also identified as CP-18821) 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.
Clinical Data
male
Markers
positive for epithelial marker pan-cytokeratin (immunocytochemistry)(verified at ATCC)
negative for gastric mucin (CLH2) (immunocytochemistry)(verified at ATCC)
Genes Expressed
positive for epithelial marker pan-cytokeratin (immunocytochemistry)(verified at ATCC),negative for gastric mucin (CLH2) (immunocytochemistry)(verified at ATCC)
Comments

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.

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. In addition, it has been verified that no gross changes are observed in karyotype and morphology during the first 10 population doublings.
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)
  • 1 nM cholera toxin
  • 20 mg/L adenine
  • 140 µg/ml BPE (Bovine Pituitary Extract)
  • 0.1% ITS [Insulin-Transferrin-Sodium Selenite Supplement(Sigma, I1884)]
  • 4 mM glutamine
  • fetal bovine serum to a final concentration of 5%
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 1 X 104 to 2 X 104 viable cells/cm2 is recommended.
  6. Incubate cultures at 37.0°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 1:5 is recommended.
Medium renewal: every 3 to 4 days
Cryopreservation
Freeze medium: RPMI-1640 Medium, 80%; fetal bovine serum, 10%; DMSO, 10%
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Temperature: 37.0°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
STR Profile
CSF1PO: 12
D13S317: 12
D16S539: 10, 13
D5S818: 9, 12
D7S820: 10, 11
THO1: 9, 9.3
TPOX: 8
vWA: 16
Amelogenin: X (Note: LOH of Y)
Population Doubling Level (PDL) >15 PDLs post cryopreservation recovery
Population Doubling Time approximately 39 hours
Name of Depositor B. Reid
Passage History
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.
Year of Origin April 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

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

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

Basic Documentation
Product Sheet
Product Sheet
Certificate of Analysis
Certificate of Analysis
MSDS
MSDS
Other Documentation
Other Document
Cell Micrograph
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

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

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