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Human mammary epithelium, HME1 cell line, was derived from a 53-year-old patient undergoing reduction mammoplasty surgery (no history of breast cancer). 

The HME1 cells were immortalized by infection with the retrovirus pBabepuro+hTERT vector and cultured in complete growth medium containing puromycin until stable clones were selected. 

Positive for the cytokeratin epithelial marker (immunocytochemistry) and for the Mucin 1, transmembrane (MUC1) protein (Homo sapiens; detection by flow cytometry). 

1. Remove and discard culture medium.
2. Add 2.0 to 3.0 mL of 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to the flask and observe cells under an inverted microscope until the cell layer is dispersed (usually within 5 to 15 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
3. To remove trypsin-EDTA solution, add 2.0 to 3.0 mL of 0.1% SoybeanTrypsin Inhibitor solution and aspirate cells by gently pipetting.
4. Transfer cell suspension to a 15 mL centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
5. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 7 x 10³ to 9x 10³ viable cells/cm²  is recommended. Subculture cultures when they reach a cell concentration between 4 x 10⁴ and 6 x 10⁴ cells/cm².
6. Incubate cultures at 37°C.

Temperature: 37°C
Atmosphere: Air, 95%; Carbon dioxide (CO₂), 5%

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Morgenstern JP, Land H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18: 3587-3596, 1990. PubMed: 2194165

Shay JW, et al. E6 of human papillomavirus type 16 can overcome the M1 stage of immortalization in human mammary epithelial cells but not in human fibroblasts. Oncogene 8: 1407-1413, 1993. PubMed: 8389027

Gollahon LS, Shay JW. Immortalization of human mammary epithelial cells transfected with mutant p53 (273his). Oncogene 12: 715-725, 1996. PubMed: 8632893

Van Der Haegen BA, Shay JW. Immortalization of human mammary epithelial cells by SV40 large T-antigen involves a two step mechanism. In Vitro Cell. Dev. Biol. 29: 180-182, 1993. PubMed: 8463179

Caputo JL. Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988

Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 4th edition. New York: Wiley Liss; 2000. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 10.