HBEC3-KT (ATCC® CRL-4051)

Organism: Homo sapiens, human  /  Cell Type: epithelial  /  Tissue: lung, bronchial  /  Disease: normal

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Organism Homo sapiens, human
Tissue lung, bronchial
Cell Type epithelial
Product Format frozen
Morphology epithelial, packed cuboidal
Culture Properties adherent
Biosafety Level 2  [Cells immortalized by CDK4 and hTERT and contain SV40 promoter sequences]
Disease normal
Age 65
Gender female
Applications

HBEC3-KT cells are normal human bronchial epithelial cells immortalized with CDK4 and hTERT.  The immortalized HBEC3-KT cells do not form colonies in soft agar, nor do they form tumors in mice (RefRamirez RD, Sheridan S, Girard L, et al. Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res. 64(24):9027-9034, 2004. PubMed: 15604268).  Multiple oncogenic changes (K-RAS(V12), p53 knockdown, mutant EGFRs) are not sufficient to confer a full malignant phenotype on HBEC3-KT. These additional genetic changes, commonly found in human lung cancer, progress the HBEC3-KT cells partially, but not completely, toward malignancy (RefSato M, et al. Multiple oncogenic changes (K-RAS(V12), p53 knockdown, mutant EGFRs, p16 bypass, telomerase) are not sufficient to confer a full malignant phenotype on human bronchial epithelial cells. Cancer Res. 66(4): 2116-2128, 2006. PubMed: 16489012). 

Extended lifespan (RefRamirez RD, Sheridan S, Girard L, et al. Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res. 64(24):9027-9034, 2004. PubMed: 15604268), multi-potent differentiation capacity in three-demensional models (RefDelgado O, et al. Multipotent capacity of immortalized human bronchial epithelial cells. PLoS ONE. 6(7): e22023, 2011. PubMed: 21760947), and drug-sensitivity tests (paclitaxel, carboplatin, pemetrexed, cisplatin, gemcitabine, etc.) have been reported for the HBEC3-KT cells (RefSato M, et al. Human lung epithelial cells progressed to malignancy through specific oncogenic manipulations. Mol. Cancer Res. 11(6): 638-650, 2013. PubMed: 23449933).

Storage Conditions liquid nitrogen vapor phase
Karyotype Cytogenetic analysis was performed on G-banded metaphase cells from the human cell line HBEC3-KT, and demonstrated mixed population of clones with near-diploid or tetraploid abnormal female karyotypes. Unbalanced translocations der(16)t(5;16)(q11.2;q24) and der(13)t(8;13)(q13.1;p11.1) as well as an extra copy of chromosome 20 with additional translocation +add(20)(q13.3) are observed in these clones. These aberrations result in partial trisomies of chromosome 5, 8 or 20 which have been found in a previous aCGH microarray analysis of HBEC3-KT (PMID: 15604268)
Images
Derivation

The HBEC3-KT cell line was established by infecting primary human bronchial epithelial cell culture with human telomerase (hTERT) and mouse cyclin dependent kinase 4 (CDK4) expressing retrovirus constructs and selecting with Puromycin and G418 as described in PMID: 15604268 (RefRamirez RD, Sheridan S, Girard L, et al. Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res. 64(24):9027-9034, 2004. PubMed: 15604268).

Clinical Data female
65 years
Antigen Expression Positive for p63 (TP63) and Clara cell 10 (CC10) protein
Complete Growth Medium Airway Epithelial Cell Basal Medium (ATCC PCS-300-030) suplemented with Bronchial Epithelial Cell Growth Kit (ATCC PCS-300-040)
Subculturing Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation solutions for culture vessels of other sizes.

Subculture when the culture is about 70-80% confluent.

  1. Remove and discard spent medium.
  2. Briefly rinse the cells with Dulbecco's Phosphate Buffered Saline (DPBS, ATCC 30-2200), 1 mL / 25 cm2 and discard rinse solution.
  3. Add Trypsin-EDTA, at 1 mL / 25 cm2, for Primary Cells (ATCC PCS-999-003) to the flask. Incubate at 37°C for 4-6 min (until 90% of the cells have detached).
  4. Rapt flask gently to ensure cells are detached.  Add 2% FBS in DPBS at 1 mL / 25 cm2 to neutralize the trypsin.
  5. Centrifuge cells at 1000rpm for 5 min at room temperature.
  6. Remove supernatant. Resuspend pellet in 6.0 to 8.0 mL Complete Growth Medium.
  7. Count cells, and seed 4.0 x 103 to 6.0 x 103 viable cells/cm2 to new culture vessels.
Medium Renewal: Every 2-3 days.

Note: Cells are sensitive to FBS solution.

Cryopreservation 80% complete growth media, 10% DMSO, 10% FBS
Culture Conditions Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C
STR Profile D5S818:         12   
D13S317:       12, 13
D7S820:         8, 12
D16S539:       9, 12
vWA:              15, 17 
Amelogenin:     X
TPOX:              11, 12
CSF1PO:         10, 11
TH01:               9.3
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 John Minna, Shelley Sheridan
References

Ramirez RD, Sheridan S, Girard L, et al. Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res. 64(24):9027-9034, 2004. PubMed: 15604268

Delgado O, et al. Multipotent capacity of immortalized human bronchial epithelial cells. PLoS ONE. 6(7): e22023, 2011. PubMed: 21760947

Sato M, et al. Human lung epithelial cells progressed to malignancy through specific oncogenic manipulations. Mol. Cancer Res. 11(6): 638-650, 2013. PubMed: 23449933

Sato M, et al. Multiple oncogenic changes (K-RAS(V12), p53 knockdown, mutant EGFRs, p16 bypass, telomerase) are not sufficient to confer a full malignant phenotype on human bronchial epithelial cells. Cancer Res. 66(4): 2116-2128, 2006. PubMed: 16489012

Vaughan MB, et al. A three-dimensional model of differentiation of immortalized human bronchial epithelial cells. Differentiation. 74(4):141-148, 2006. PubMed: 16683984

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
FAQ's
  1. HBEC3-KT Seeding Density
    The optimal seeding density for HBEC3-KT (ATCC® CRL-4051 ™) is 4,000 to 6,000 cells/cm
    Date Updated: 6/17/2014
  2. When to subculture HBEC3-KT cells
    HBEC3-KT (ATCC® CRL-4051 ™) should be subcultured when the cells reach 70% to 80% confluence, usually 3-4 days.
    Date Updated: 6/17/2014
  3. Recommended HBEC3-KT Growth Medium
    The recommended growth medium for HBEC3-KT (ATCC® CRL-4051 ™) is Airway Epithelial Cell Basal Medium (ATCC® PCS-300-030 ™) supplemented with the Bronchial Epithelial Cell Grow...
    Date Updated: 6/17/2014
  4. Freezing HBEC3-KT Cells
    HBEC3-KT (ATCC® CRL-4051 ™) cells should be frozen in 80% complete growth medium:10% FBS:10%DMSO.  Store the cryopreserved cells in the vapor phase of liquid nitrogen.
    Date Updated: 6/17/2014
  5. Using Antibiotics with HBEC3-KT

    Antibiotics are not recommended as they can mask contamination.


    Date Updated: 6/17/2014
  6. Thawing HBEC3-KT Cells


    Date Updated: 6/17/2014

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

Ramirez RD, Sheridan S, Girard L, et al. Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res. 64(24):9027-9034, 2004. PubMed: 15604268

Delgado O, et al. Multipotent capacity of immortalized human bronchial epithelial cells. PLoS ONE. 6(7): e22023, 2011. PubMed: 21760947

Sato M, et al. Human lung epithelial cells progressed to malignancy through specific oncogenic manipulations. Mol. Cancer Res. 11(6): 638-650, 2013. PubMed: 23449933

Sato M, et al. Multiple oncogenic changes (K-RAS(V12), p53 knockdown, mutant EGFRs, p16 bypass, telomerase) are not sufficient to confer a full malignant phenotype on human bronchial epithelial cells. Cancer Res. 66(4): 2116-2128, 2006. PubMed: 16489012

Vaughan MB, et al. A three-dimensional model of differentiation of immortalized human bronchial epithelial cells. Differentiation. 74(4):141-148, 2006. PubMed: 16683984