產(chǎn)品名稱 |
CP-A (KR-42421) |
商品貨號 |
B229006 |
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 [Cells contain SV40 viral DNA sequences]
Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country. |
Disease |
Non-dysplastic metaplasia |
Age |
Adult |
Gender |
Male |
Applications |
This well-characterized pre-malignant culture represents a unique opportunity to study factors that are important in both cancer progression and hTERT immortalization. |
Storage Conditions |
Liquid nitrogen vapor phase |
Karyotype |
This is a near-diploid cell line of male origin in which 2 sub-clones make up the majority of the cell population. One clone containing i(8)(q10) and trisomy 20 and the other containing der(1)t(1;18)(q10;q10), i(8)(q10), der(13)t(13;22)(q10;q10) and trisomy 20. The remaining population is generally made up of cells with non-clonal aberrations that were derived from the two major clones. Also, the non-clonal cell population may increase at high passages. |
Images |
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Derivation |
The Barrett's esophagus cell line, CP-A (also identified as KR-42421 or QhTERT), was derived from an endoscopic biopsy specimen obtained from a region of non-dysplastic metaplasia and transduced with the retroviral expression vector, pLXSN-hTERT, to create an immortalized cell line. |
Antigen Expression |
Positive for epithelial marker pan-cytokeratin (immunocytochemistry) (verified at ATCC)
Negative for gastric mucin (CHL2) (immunocytochemistry) (verified at ATCC) |
Comments |
This cell line shows increased telomerase activity and long telomeres of about 12 kb as assessed by terminal restriction fragment lengths (TRF) analysis. Morphologically, the cells are similar to early passage cultures: smaller cells with a larger 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.
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Complete Growth Medium |
The base medium for this cell line is MCDB-153. For complete growth medium, add the following components to the base medium:
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0.4 µg/ml hydrocortisone
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20 ng/ml recombinant human epidermal growth factor
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8.4 µg/L cholera toxin
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20 mg/L adenine
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140 µg/ml bovine pituitary extract
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1x ITS Supplement (Sigma; I1884)
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4 mM glutamine
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fetal bovine serum to a final concentration of 5%
To prepare Cholera toxin (Stock 100 µg/mL): 0.5 mg Cholera toxin (Sigma C8052) + 5 mL dH20. Add 84 µL of this 100 µg/mL stock solution to 1L of MCDB-153 base medium. Do not filter the complete medium. |
Subculturing |
Volumes used in this protocol are for 75 cm 2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
- Remove and discard culture medium.
- 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 37.0°C to facilitate dispersal.
- Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
- Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes. Discard supernatant.
- 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.
- Incubate cultures at 37°C. Subculture when cells reach a concentration between 8 X 104 and 1 X 105 cells/cm2.
Subcultivation ratio: A subcultivation ratio of 1:3 to 1:5 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 |
Amelogenin: X,Y CSF1PO:12 D13S317:12,14 D16S539:12,13 D5S818:11,12 D7S820:7,8 THO1: 7,9.3 TPOX: 8,9 vWA: 17 |
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 |
August 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
Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.
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