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CuFi-5 (ATCC® CRL-4016™)

Organism: Homo sapiens, human  /  Cell Type: Epithelial cells immortalized with hTERT and HPV-16 E6/E7-LXSN  /  Tissue: Lung; bronchus  /  Disease: Cystic fibrosis

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Organism Homo sapiens, human
Tissue Lung; bronchus
Cell Type Epithelial cells immortalized with hTERT and HPV-16 E6/E7-LXSN
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 32 years
Gender Male
Storage Conditions Liquid nitrogen vapor phase
Karyotype This is a near-diploid cell line of male origin in which the most consistent karyotypic aberrations are trisomy of chromosomes 5 and 20. Other non-clonal aberrations were found at early passage, but the karyology tended to stabilize within several passages.
Images ATCC CRL-4016 Micrograph
Derivation
Human airway epithelial (HAE) cell line, CuFi-5, was derived from lung of a 32-year-old patient with cystic fibrosis by retroviral infection with hTERT and HPV-16E6/E7. 
Antigen Expression
Antigen expression: This cell line is positive for the epithelial marker pan-cytokeratin as assessed by immunocytochemistry.
Comments

CuFi-5 cells are homozygous for the delta F508 cystic fibrosis-causing mutation (delta F508/delta F508).

The cells do not undergo growth arrest in cell culture due to exogenous expression of the telomerase and E6/E7 genes.

Other hTERT-immortalized cell lines derived from cystic fibrosis HAE are also available from ATCC as ATCC CRL-4013 (CuFi-1), ATCC CRL-4015 (CuFi-4), and ATCC CRL-4017 (CuFi-6).
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.
  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 104 to 2 x 104 viable cells/cm2 is recommended.
  6. Incubate cultures at 37°C.
  7. Subculture when cell concentration is between 3 x 104 and 4 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,Y
CSF1PO: 10,14
D13S317: 11,13
D16S539: 11,13
D5S818: 11,12
D7S820:11,12
THO1: 7
TPOX: 8,10
vWA: 16,17
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

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

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:

  • For-profit customers must obtain a research use license agreement from the Contributor prior to shipment.
  • 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.
Basic Documentation
Product Sheet
Product Sheet
Certificate of Analysis
Certificate of Analysis
SDS
SDS
Other Documentation
Other Document
Cell Micrograph
Restrictions

For commercial accounts, this cell line is only distributed under the terms of a fully signed and executed ATCC® Material Transfer Agreement and Addendum. If the commercial account is screening per completed Addendum, the recipient will be required to pay a Screening Fee (ATCC® ACS-2103™) of $4000.00.

Screening Use is defined as use of Biological Material in small molecule and biologic drug discovery, including initial target identification and validation, assay development, high throughput screening, hit identification, lead optimization, and selection of candidates for clinical development.

If the commercial account is not screening per the completed Addendum, the recipient will not be required to pay a Screening Fee.
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

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

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.