CuFi-6細(xì)胞

ATCC Number： CRL-4017?

相關(guān)**： 囊腫性纖維化

器官來源： 肺

細(xì)胞形態(tài)： 上皮樣

組織來源： bronchus

生長狀態(tài)： 貼壁生長

年限： 20 years

運(yùn)輸方式： 凍存運(yùn)輸

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

Designations: CuFi-6

CuFi-6細(xì)胞Depositors: AJ Klingelhutz

Biosafety Level: 2

Shipped: frozen

Medium & Serum: See Propagation

Growth Properties: adherent

Organism: Homo sapiens

Morphology: epithelial

Source: Organ: lung

Tissue: bronchus

Disease: cystic fibrosis

Cell Type: epithelial immortalized with hTERT and HPV-16 E6/E7-LXSN

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Isolation: Isolation date: 2001

Applications: 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.

Human airway epithelial (HAE) cell line, CuFi-6, was derived from lung of a 20-year-old patient with cystic fibrosis by retroviral infection with HPV-16 E6/E7-LXSN. Consequently, the cells do not undergo growth arrest in cell culture due to exogenous expression of the telomerase and E6/E7 genes.

Another hTERT-immortalized cell line derived from cystic fibrosis HAE is available as ATCC CRL-4013 (CuFi-1).

Antigen Expression: positive for epithelial marker pan-cytokeratin (immunocytochemistry)

DNA Profile (STR): Amelogenin: XY

CSF1PO: 11

D13S317: 12

D16S539: 11,12

D5S818: 9,12

CuFi-6細(xì)胞D7S820:8,11

THO1: 6

TPOX: 8,10

vWA: 17,19

Cytogenetic Analysis: This is a near-diploid cell line of male origin. The most consistent karyotypic aberrations include trisomy of chromosome 5 and 20, monosomy of chromosome 15 or 16, and loss of the Y chromosome. Additionally, the polyploidy rate may increase slightly at high passage.

Age: 20 years

Gender: male

Comments: Human airway epithelial (HAE) cell line, CuFi-6, was derived from lung of a 20-year-old patient with cystic fibrosis by retroviral infection with HPV-16 E6/E7-LXSN. Consequently, the cells do not undergo growth arrest in cell culture due to exogenous expression of the telomerase and E6/E7 genes. [22566]

CuFi-6 cells are homozygous for the delta F508 cystic fibrosis-causing mutation (delta F508/delta F508). [92666]

Another hTERT-immortalized cell line derived from cystic fibrosis HAE is available as ATCC CRL-4013 (CuFi-1).

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: These cells are grown in a serum-free medium: BEGM (Bronchial Epithelial Growth Medium, Serum-free) from Lonza (BEGM Bullet Kit; CC-3170) made of BEBM basal medium and SingleQuot additives (ATCC does not use gentamycin-amphotericin B) supplemented with 50 ?g/ml G-418.

Temperature: 37.0°C

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

Subculturing: Protocol:

Note: The culture flasks should be pre-coated with 60μg/ml solution of Human Placental Collagen Type IV. (Sigma, Cat. No. C-7521) at least 18 hours in advance then air-dried and rinsed 2-3 times with Dulbecco's Phosphate Buffered Saline.

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 Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).

Note: CuFi-6細(xì)胞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.

To remove Trypsin-EDTA solution, add 2.0 to 3.0 ml of 1% FBS in Dulbecco's Phosphate buffered Saline 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 5 x 103 to 1 x 104 viable cells/cm2 is recommended.

Incubate cultures at 37°C.

Subculture when cell concentration is between 2 x 104 and 3 x 104 cells/cm2.

Subcultivation ratio: A subcultivation ratio of 1:2 to 1:3 is recommended.

Medium renewal: every 3 to 4 days

Preservation: Freeze medium BEGM supplemented with 10% (v/v) DMSO and 30% (v/v) fetal bovine serum

Storage temperature: liquid nitrogen vapor phase

Doubling Time: about 64 hours

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

Recommended serum: ATCC 30-2020

Phosphate-buffered saline: ATCC 30-2200

Cell culture tested DMSO: ATCC 4-X

References: 22566: Halbert CL, et al. The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. J. Virol. 65: 473-478, 1991. PubMed: 1845902

47354: Bodnar AG, et al. CuFi-6細(xì)胞Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349-352, 1998. PubMed: 9454332

92666: Zabner J, et al. Development of cystic fibrosis and noncystic fibrosis airway cell lines . Am. J. Physiol. Lung Cell Mol Physiol. 284: L844-L854, 2003. PubMed: 12676769

92667: Kiyono T, et al. Both Rb/p161NK4a inactivation and telomerase activity are required to immortalize human epithelial cells. Nature 396: 84-88, 1998. PubMed: 9817205