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CuFi-4

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編號:B224506

品牌:Mingzhoubio

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產品名稱

CuFi-4

商品貨號

B224506

Organism

Homo sapiens, human

Tissue

Lung; bronchus

培養基

Cell Type

Epithelial cells immortalized with hTERT, HPV-16 E6/E7-LXSN, and pBABE-Hyg-TERT

Product Format

frozen

Morphology

Epithelial-like

Culture Properties

Adherent

Biosafety Level

2 [Cells contain SV40 and HPV 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

Cystic fibrosis

Age

33 years

Gender

Female

Applications

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

Storage Conditions

Liquid nitrogen vapor phase

Karyotype

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.

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Derivation

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.

Antigen Expression

Antigen expression: This cell line is positive for epithelial marker pan-cytokeratin as assessed by immunocytochemistry.

Comments

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.

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 μg/ml G-418.

Subculturing

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 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 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

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: BEGM with 30% FBS and 10% DMSO

Storage temperature: liquid nitrogen vapor phase

Culture Conditions

Temperature: 37°C

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

STR Profile

Amelogenin: X

CSF1PO: 13

D13S317: 11,12

D16S539: 12,13

D5S818: 11,13

D7S820: 10,12

THO1: 7

TPOX: 8,11

vWA: 17,18

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

AJ Klingelhutz

Year of Origin

2001

References

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

Bodnar AG, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349-352, 1998. PubMed: 9454332

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

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

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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