CP-A (KR-42421)細(xì)胞

組織來源： epithelium

生長狀態(tài)： 貼壁生長

器官來源： 食道

細(xì)胞形態(tài)： 上皮樣

細(xì)胞類型： 其他細(xì)胞類型

數(shù)量： 大量

是否是腫瘤細(xì)胞： 0

物種來源： 人

ATCC Number： CRL-4027?

相關(guān)**： Barrett食管

運(yùn)輸方式： 凍存運(yùn)輸

Designations: CP-A (KR-42421)

Depositors: B Reid

CP-A (KR-42421)細(xì)胞Biosafety Level: 2

Shipped: frozen

Medium & Serum: See Propagation

Growth Properties: adherent

Organism: Homo sapiens

Morphology: epithelial-like

Source: Organ: esophagus

Tissue: epithelium

Cell Type: non-dysplastic metaplasia

Disease: Barrett's esophagus

Immortalization method: hTERT expression

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Isolation: Isolation date: August 1995

Applications: CP-A (KR-42421)細(xì)胞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.

This cell line shows increased telomerase activity and long telomeres of about 12 kb as assessed by terminal restriction fragment lengths (TRF) analysis.

In addition, it has been verified that no gross changes are observed in karyotype and morphology during the first 10 population doublings.

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.

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 )

DNA Profile (STR): 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

Cytogenetic Analysis: 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.

Gender: male

Comments: CP-A (KR-42421)細(xì)胞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.

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.

This well-characterized pre-malignant culture represents a unique opportunity to study factors that are important in both cancer progression and hTERT immortalization. [16173160]

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.

Propagation: 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 ?g/ml hydrocortisone

20 ng/ml recombinant human EGF (Epidermal Growth Factor)

1 nM cholera toxin

20 mg/L adenine

140 ?g/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%

Temperature: 37.0°C

Atmosphere: air, 95%; carbon dioxide (CO2), 5%

Subculturing: Protocol: CP-A (KR-42421)細(xì)胞Volumes used in this protocol are for 75 cm2 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 xg 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 37.0°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

Preservation: Freeze medium: RPMI-1640 Medium, 80%; fetal bovine serum, 10%; DMSO, 10%

Storage temperature: liquid nitrogen vapor phase

Doubling Time: approximately 34 hours

Related Products: Recommended base medium for freezing (without the additional serum described under Freeze Medium): ATCC 30-2001

Recommended serum: ATCC 30-2020

0.25% (w/v) Trypsin - 0.53mM EDTA in Hank's BSS (w/o Ca++, Mg ++): ATCC 30-2101

Cell culture tested DMSO: ATCC 4-X

Erythrosin B vital stain solution: ATCC 30-2404

References: 16173160: 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

16173161: 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

16173615: 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

16173616: Maley CC, et al. Genetic clonal diversity predicts progression to esophageal adenocarcinoma. Nat. Genet. 38(4): 468-473, 2006. PubMed: 16565718