HBEC3-KT

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).

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).

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.

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.

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.

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.

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