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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).

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.

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