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HBEC3-KT

CRL-4051

Product category
Human cells
Product type
hTERT-immortalized cell
Organism
Homo sapiens, human
Cell type
epithelial cell
Morphology
epithelial, packed cuboidal
Tissue
Lung; Bronchus
Disease
Normal
Applications
3D cell culture
Drug development
High-throughput screening
Toxicology
Product format
Frozen
Storage conditions
Vapor phase of liquid nitrogen
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Documentation

Biosafety Icon BSL 2

ATCC determines the biosafety level of a material based on our risk assessment as guided by the current edition of Biosafety in Microbiological and Biomedical Laboratories (BMBL), U.S. Department of Health and Human Services. It is your responsibility to understand the hazards associated with the material per your organization’s policies and procedures as well as any other applicable regulations as enforced by your local or national agencies.

Cells immortalized by CDK5

Cells immortalized by hTERT

Cells contain SV40 promoter sequences

ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.

Required Products

These products are vital for the proper use of this item and have been confirmed as effective in supporting functionality. If you use alternative products, the quality and effectiveness of the item may be affected.

Airway Epithelial Cell Basal Medium

PCS-300-030

Price: $55.00 ea

Bronchial Epithelial Cell Growth Kit

PCS-300-040

Price: $80.00 ea

Dulbecco's Phosphate Buffered Saline (D-PBS), 1X

30-2200

Price: $16.00 ea

Trypsin-EDTA for Primary Cells

PCS-999-003

Price: $28.00 ea

Dimethylsulfoxide (DMSO)

4-X

Price: $49.00 ea

Detailed product information

General

Specific 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).

Characteristics

Growth properties
Adherent
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).

Age
65 years
Gender
Female
Immortalization method
hTERT expression and mouse cyclin dependent kinase 4 (CDK4) expressing retrovirus
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)
Antigen expression
Positive for p63 (TP63) and Clara cell 10 (CC10) protein

Handling information

Unpacking and storage instructions
  1. Check all containers for leakage or breakage.
  2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.
Complete medium
Airway Epithelial Cell Basal Medium (ATCC PCS-300-030) suplemented with Bronchial Epithelial Cell Growth Kit (ATCC PCS-300-040)
Temperature
37°C
Atmosphere
95% Air, 5% CO2
Handling procedure
To ensure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt.
If storage of the frozen culture is necessary upon arrival, store the vial in liquid nitrogen vapor phase and NOT
at –70°C. Storage at –70°C will result in loss of viability.
  1. Prepare a 25 cm2 or a 75 cm2 culture flask containing the recommended complete culture medium. Prior to the addition of the vial contents, the vessel containing the growth medium should be placed in the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6) and to avoid excessive alkalinity of the medium during recovery of the cells.
  2. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
  3. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All operations from this point on should be carried out under strict aseptic conditions.
  4. Transfer the vial contents to a centrifuge tube containing 3.0 mL of complete culture medium, mix gently, then slowly add an additional 7.0 mL of complete growth medium and centrifuge the cell suspension at approximately 1000 rpm for 5 minutes at room temperature.
  5. Discard the supernatant (ENSURING THAT AS MUCH OF THE FBS/DMSO IS REMOVED AS THESE CELLS ARE SENSITIVE TO IT) and resuspend the cells in 3 mL of fresh growth medium. Count the cells and seed at recommended seeding density.  
  6. Incubate the culture at 37°C in a suitable incubator.
Subculturing procedure
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. tap 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: The cells are sensitive to DMSO and FBS. Ensure that as much as possible is removed after centrifugation, and before seeding into the recommended culture medium.

Reagents for cryopreservation
Complete growth medium supplemented with 10% (v/v) FBS and 10% (v/v) DMSO (ATCC 4-X).

Quality control specifications

Population doubling capacity
≥ 15 in complete growth medium
STR profiling
D5S818:         12   
D13S317:       12, 13
D7S820:         8, 12
D16S539:       9, 12
vWA:              15, 17 

History

Depositors
John Minna, Shelley Sheridan

Legal disclaimers

Intended use
This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use.
Warranty

The product is provided 'AS IS' and the viability of ATCC® products is warranted for 30 days from the date of shipment, provided that the customer has stored and handled the product according to the information included on the product information sheet, website, and Certificate of Analysis. For living cultures, ATCC lists the media formulation and reagents that have been found to be effective for the product. While other unspecified media and reagents may also produce satisfactory results, a change in the ATCC and/or depositor-recommended protocols may affect the recovery, growth, and/or function of the product. If an alternative medium formulation or reagent is used, the ATCC warranty for viability is no longer valid.  Except as expressly set forth herein, no other warranties of any kind are provided, express or implied, including, but not limited to, any implied warranties of merchantability, fitness for a particular purpose, manufacture according to cGMP standards, typicality, safety, accuracy, and/or noninfringement.

Disclaimers

This product is intended for laboratory research use only. It is not intended for any animal or human therapeutic use, any human or animal consumption, or any diagnostic use. Any proposed commercial use is prohibited without a license from ATCC.

While ATCC uses reasonable efforts to include accurate and up-to-date information on this product sheet, ATCC makes no warranties or representations as to its accuracy. Citations from scientific literature and patents are provided for informational purposes only. ATCC does not warrant that such information has been confirmed to be accurate or complete and the customer bears the sole responsibility of confirming the accuracy and completeness of any such information.

This product is sent on the condition that the customer is responsible for and assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, and use of the ATCC product including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. As a condition of receiving the material, the customer agrees that any activity undertaken with the ATCC product and any progeny or modifications will be conducted in compliance with all applicable laws, regulations, and guidelines. This product is provided 'AS IS' with no representations or warranties whatsoever except as expressly set forth herein and in no event shall ATCC, its parents, subsidiaries, directors, officers, agents, employees, assigns, successors, and affiliates be liable for indirect, special, incidental, or consequential damages of any kind in connection with or arising out of the customer's use of the product. While reasonable effort is made to ensure authenticity and reliability of materials on deposit, ATCC is not liable for damages arising from the misidentification or misrepresentation of such materials.

Please see the material transfer agreement (MTA) for further details regarding the use of this product. The MTA is available at www.atcc.org.

Permits & Restrictions

Material Transfer Agreement Addendum for Screening Applications

For-profit organizations
For every order of this item, you must provide a signed Material Transfer Agreement Addendum for Screening Applications. We cannot ship this item until we receive this addendum. The person signing the addendum as the principal investigator must match the end user as listed on the applicable sales order for the item.

Email the signed addendum to [email protected] with a reference to both your account and sales order numbers. Once received, your addendum will be reviewed, and this item will be released for shipment if all requirements are met. Additional fees may apply if this product is being used for a screening use (ATCC ACS-2103F), and these fees will be applied after your order is confirmed. If you need assistance with your order, please contact our Customer Care team or your applicable distributor.

Import Permit for the State of Hawaii

If shipping to the U.S. state of Hawaii, you must provide either an import permit or documentation stating that an import permit is not required. We cannot ship this item until we receive this documentation. Contact the Hawaii Department of Agriculture (HDOA), Plant Industry Division, Plant Quarantine Branch to determine if an import permit is required.

MORE INFORMATION ABOUT PERMITS AND RESTRICTIONS

References

Curated Citations

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

Frequently Asked Questions

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Telephone

Telephone

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Outside the US
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