CuFi-1 (ATCC® CRL-4013)

Organism: Homo sapiens, human  /  Cell Type: Epithelial cells immortalized E6/E7 and hTERT expression  /  Tissue: Lung; epithelium, bronchus  /  Disease: Cystic fibrosis

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Organism Homo sapiens, human
Tissue Lung; epithelium, bronchus
Cell Type Epithelial cells immortalized E6/E7 and hTERT expression
Product Format frozen
Morphology Epithelial-like
Culture Properties Adherent
Biosafety Level 2 [cells contain SV40 and HPV viral DNA sequences]
Disease Cystic fibrosis
Age 14 years
Gender Female
Applications
These cell lines may be useful models for studying ion physiology, therapeutic interventions for cystic fibrosis and innate immunity [Pubmed: 12676769].
Storage Conditions Liquid nitrogen vapor phase
Karyotype This is a near-diploid human cell line of female origin with a modal chromosome count of 46 and a polyploidy rate of 27%. There were two copies of a karyotypically normal X-chromosome present in most of the cells. Overall, some of the cells contained chromosomal abnormalities, with most consistent being trisomy 20.
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Derivation
Human airway epithelial (HAE) cell line, CuFi-1, was derived from lung of a 14-year-old patient with cystic fibrosis by dual retroviral infection with HPV-16E6/E7-LXSN [Pubmed: 1845902] and hTERT-LXSN [Pubmed: 9817205]. 
Comments

The cells do not undergo growth arrest in cell culture due to exogenous expression of the telomerase and E6/E7 genes. CuFi-1 cells are homozygous for the delta F508 cystic fibrosis-causing mutation (delta F508/delta F508). 

Another hTERT-immortalized cell line, derived from normal HAE is also available as ATCC CRL-4011 (NuLi-1). Both cell lines, when seeded on semipermeable filters and grown at the air-liquid interface, are capable of forming polarized differentiated epithelia that exhibit transepithelial resistance and maintain the ion channel physiology expected of each genotype. 

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 60 μg/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.

  1. Remove and discard culture medium.
  2. 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.
  3. 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.
  4. Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes. Discard supernatant.
  5. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 1 x 10to 3 x 103 viable cells/cm2 is recommended.
  6. Incubate cultures at 37°C.
  7. Subculture when cell concentration is between 1 x 104 and 2 x 104 cells/cm2.
Subcultivation Ratio: 1:5
Medium Renewal: Every 2-3 days (do not exceed 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
Atmosphere: 5% CO2 in air recommended
Temperature: 37°C
STR Profile
Amelogenin: X
CSF1PO: 12
D13S317: 11,13
D16S539: 11,14
D5S818: 12
D7S820: 8,11
THO1: 6,9.3
TPOX: 8,11
vWA: 17,20
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 2000
References

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

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

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

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.

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
  • License agreement required for commercial customer uses.
  • This material is distributed for research purposes only. A signed addendum to the ATCC Material Transfer Agreement must be sent to ATCC in advance of shipment.
Basic Documentation
Other Documentation
Restrictions

This material is subject to claims under U.S. Patent Nos. 6,261,836 and 6,337,200, other pending patent applications, and foreign counterparts thereof. It is required that either the Addendum for Commercial and For-Profit Organizations or the Addendum for Noncommercial and Academic Organizations, as appropriate, be signed and returned to ATCC before shipment.

References

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

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

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

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.