CuFi-4細(xì)胞

器官來源： 肺

組織來源： bronchus

ATCC Number： CRL-4015?

相關(guān)**： 囊腫性纖維化

細(xì)胞類型： 其他細(xì)胞類型

是否是腫瘤細(xì)胞： 0

物種來源： 人

細(xì)胞形態(tài)： 上皮樣

運(yùn)輸方式： 凍存運(yùn)輸

生長(zhǎng)狀態(tài)： 貼壁生長(zhǎng)

年限： 33 years

數(shù)量： 大量

Designations: CuFi-4

Depositors: AJ Klingelhutz

CuFi-4細(xì)胞Biosafety Level: 2

Shipped: frozen

Medium & Serum: See Propagation

Growth Properties: adherent

Organism: Homo sapiens

Morphology: epithelial-like

Source: Organ: lung

Tissue: bronchus

Disease: cystic fibrosis

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

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Isolation: Isolation date: 2001

Applications: CuFi-4細(xì)胞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-4, was derived from lung of a 33-year-old patient with cystic fibrosis by dual retroviral infection with HPV-16E6/E7-LXSN and pBabe-hygro-hTERT. Consequently, the cells do not undergo growth arrest in cell culture due to exogenous expression of the telomerase and E6/E7 genes.

This cell line may be a useful model for studying ion channel physiology, therapeutic interventions for cystic fibrosis and innate immunity.

CuFi-4 cells are heterozygous for the cystic fibrosis-causing mutations delta F508/G551D. When seeded on semi-permeable filters and cultured at an air-liquid interface, they are capable of forming polarized, differentiated epithelia that exhibit transepithelial resistance and maintain the ion channel physiology expected of a bronchial epithelium.

Other hTERT-immortalized cell lines derived from cystic fibrosis HAE are also available from ATCC as ATCC CRL-4013 (CuFi-1), ATCC CRL-4016 (CuFi-5) and ATCC CRL-4017 (CuFi-6).

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

DNA Profile (STR): Amelogenin: X

CSF1PO: 13

D13S317: 11,12

D16S539: 12,13

D5S818: 11,13

D7S820: 10,12

THO1: 7

TPOX: 8,11

vWA: 17,18

Cytogenetic Analysis: CuFi-4細(xì)胞The karyotypes of several different passages were determined. This is a human cell line of female origin, and the ploidies range from near-diploid to near-tetraploid. The karyology seems to stabilize at higher passages in the hyperdiploid range with trisomies or tetrasomies of chromosomes 1, 5, 8, 11 and 20. Additional copies of chromosomes 5 and 20 were the most consistent aberrations found throughout all the passages and ploidies.

Age: 33 years

Gender: female

Comments: Human airway epithelial (HAE) cell line, CuFi-4, was derived from lung of a 33-year-old patient with cystic fibrosis by dual retroviral infection with HPV-16E6/E7-LXSN and pBabe-hygro-hTERT. Consequently, the cells do not undergo growth arrest in cell culture due to exogenous expression of the telomerase and E6/E7 genes. [22566]

CuFi-4 cells are heterozygous for the cystic fibrosis-causing mutations delta F508/G551D. When seeded on semi-permeable filters and cultured at an air-liquid interface, they are capable of forming polarized, differentiated epithelia that exhibit transepithelial resistance and maintain the ion channel physiology expected of a bronchial epithelium. This cell line may be a useful model for studying ion channel physiology, therapeutic interventions for cystic fibrosis and innate immunity. [92666]

Other hTERT-immortalized cell lines derived from cystic fibrosis HAE are also available from ATCC as ATCC CRL-4013 (CuFi-1), ATCC CRL-4016 (CuFi-5) and ATCC CRL-4017 (CuFi-6).

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: CuFi-4細(xì)胞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: 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 1 x 104 to 2 x 104 viable cells/cm2 is recommended.

Incubate cultures at 37°C.

Subculture when cell concentration is between 4 x 104 and 5 x 104 cells/cm2.

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

Medium renewal: every 2 to 3 days

Preservation: Freeze medium: BEGM with 30% FBS and 10% DMSO

Storage temperature: liquid nitrogen vapor phase

Doubling Time: CuFi-4細(xì)胞about 34 hours

Related Products: 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

Phosphate-buffered saline: ATCC 30-2200

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. 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