The immortalized HSAEC1-KT cells show a stable epithelial morphology and differentiated cytokeratin isoforms after over 100 population doublings, express the stem cell marker p63 and high levels of p16INK4a, and have an intact p53 checkpoint pathway (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). xa0

The HSAEC1-KT cells also exhibit characteristics of normal cells such as contact inhibition of growth and failure to form soft agar colonies or form tumors in immune compromised mice (RefKalita M, et al. Systems approaches to modeling chronic mucosal inflammation. Biomed. Res. Int. 2013: 505864, 2013. PubMed: 24228254).

TGFβ-treated HSAEC1-KT cells showed an elongated shape with markedly induced F-actin staining. This morphological change of enhanced front-rear polarity and cytoskeletal actin rearrangement are characteristic morphological changes of EMT (epithelial mesenchymal transition) (RefKalita M, et al. Systems approaches to modeling chronic mucosal inflammation. Biomed. Res. Int. 2013: 505864, 2013. PubMed: 24228254).

Cytogenetic analysis was performed on G-banded metaphase cells from the human cell line HSAEC1-KT. Several abnormal male near-diploid karyotypes are found:xa0

Clone 1 demonstrates trisomy 5 with no other aberrations. 47,XY,+5xa0

Clone 2 demonstrates trisomy 5 and 20 with no other aberrations. 48,XY,+5,+20xa0

Clone 3 demonstrates trisomy 5, an isochromosome of the long-arm of chromosome 10, resulting in three copies of the chromosome 10 long-arm and only one copy of the short-arm, and trisomy 20. 48,XY,+5,i(10)(p10),+20

Clone 4 demonstrates a deletion on the short-arm of chromosome 10 at band p10, a deletion on the short-arm of chromosome 17 at band p13, and trisomy 20. 47,XY,del(10)(p10),del(17)(p13),+20.

The HSAEC1-KT cell line was established by infecting primary human small airway epithelial cell culture with human telomerase (hTERT) and mouse cyclin dependent kinase 4 (CDK4) expressing retrovirus constructs and selecting under 250 ng/mL puromycin and 30 ug/mL 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)

To make the complete culture medium, add SAGM™ SingleQuots™ (Lonza CC-4124) which contains supplements and growth factors (BPE, Hydrocortisone, hEGF, Epinephrine, Transferrin, Insulin, Retinoic Acid, Triiodothyronine, BSA-FAF) to 500 mL bottle of SABM Basal Medium™, phenol red free basal medium (Lonza CC-3119)

1. Remove and discard spent medium.
2. Briefly rinse the cells with Dulbeccos Phosphate Buffered Saline (DPBS, ATCC 30-2200), 1 mL / 25 cm²xa0and discard rinse solution.
3. Add Trypsin-EDTA, atxa01 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. xa0Add 2% FBS in DPBS atxa01 mL / 25 cm²xa0to 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 8.0 x 10³xa0to 10.0 x 10³xa0viable cells/cm²xa0to new culture vessels.

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

Kalita M, et al. Systems approaches to modeling chronic mucosal inflammation. Biomed. Res. Int. 2013: 505864, 2013. PubMed: 24228254

Gazdar AF, Gao B, Minna JD. Lung cancer cell lines: Useless artifacts or invaluable tools for medical science? Lung Cancer 68(3): 309-318, 2010. PubMed: 20079948