In Vitro Cellular & Developmental Biology

, Volume 26, Issue 12, pp 1186–1194 | Cite as

Routine culturing of normal, dysplastic and malignant human mammary epithelial cells from small tissue samples

  • Joanne T. Emerman
  • Darcy A. Wilkinson
Rapid Communications in Cell Biology


We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca++ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca++ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely prouce sufficient numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression.

Key words

human mammary epithelial cells primary culture serum-free medium 


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  1. 1.
    Alon, Y.; Horwitz, A. T.; Biran, S., et al. Immunofluorescent characterization of fibronectin, laminin, and keratin in normal and neoplastic human mammary epithelial cells in culture and in breast tissue sections. Int. J. Tiss. Reac. 8:401–410; 1986.Google Scholar
  2. 2.
    Balakrishnan, A.; Yang, J.; Beattie, C. W., et al. Estrogen receptor in dissociated and cultured human breast fibroadenoma epithelial cells. Cancer Letters 34:233–242; 1987.PubMedCrossRefGoogle Scholar
  3. 3.
    Band, V.; Sager, R. Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types. Proc. Natl. Acad. Sci. U.S.A. 86:1249–1253; 1989.PubMedCrossRefGoogle Scholar
  4. 4.
    Biran, S.; Vlodasky, I.; Fuks, Z., et al. Growth of human mammary carcinoma cells from specimens in serum free medium on extracellular matrix. Int. J. Cancer 38:345–354; 1986.PubMedCrossRefGoogle Scholar
  5. 5.
    Briand, P. I.; Petersen, O. W.; Van Deurs, B. A new diploid nontumorigenic human breast epithelial cell line isolated and propagated in chemically defined medium. In Vitro Cell Dev. Biol. 23:181–188; 1987.PubMedGoogle Scholar
  6. 6.
    Choongkittaworn, N.; Hosick, H. L.; Jones, W. In vitro replication potential of serially passaged mammary parenchyma from mice with different reproductive histories. Mechanisms of Ageing and Development. 39:147–175; 1987.CrossRefGoogle Scholar
  7. 7.
    Connell, N. D.; Rheinwald, J. G. Regulation of the cytoskeleton in mesothelial cells: reversible loss of keratin and increase in vimentin during rapid growth in culture. Cell 34:245–253; 1983.PubMedCrossRefGoogle Scholar
  8. 8.
    Culling, C. F. A. Handbook of Histopathological and Histochemical Techniques. Third Edition. Butterworth London, England. pp. 351–363; 1975.Google Scholar
  9. 9.
    Emerman, J. E.; Fielder, E. E.; Tolcher, A. W., et al. Effects of defined medium, fetal bovine serum, and human serum on growth and chemosensitivities of human breast cancer cells in primary culture: Inference for in vitro assays. In Vitro Cell. Dev. Biol. 23:134–140; 1987.PubMedGoogle Scholar
  10. 10.
    Emerman, J. T.; Pitelka, D. R. Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In Vitro 13:316–328; 1977.PubMedCrossRefGoogle Scholar
  11. 11.
    Emerman, J. T.; Tolcher, A. W.; Rebbeck, P. M. In Vitro sensitivity testing of human breast cancer cells to hormones and chemotherapeutic agents. Cancer Chemother. Pharmacol. 26:245–249; 1990.PubMedCrossRefGoogle Scholar
  12. 12.
    Engle, L. W.; Young, N. A. Human breast carcinoma cells in continuous culture: a review. Cancer Res. 38:4327–4339; 1978.Google Scholar
  13. 13.
    Gilks, B.; Tavassoli, F. A. Coexistence of intracytoplasmic lumens and membrane-bound vesicles in an invasive carcinoma arising in a cystocarcinoma phyllodes. Ultrastruct. Pathol. 12:631–642; 1988.PubMedGoogle Scholar
  14. 14.
    Hall, L.; Henney, A.; Ralphs, D. N. L., et al. Changes in gene expression in established human mammary tumor cell lines when compared with normal breast and breast tumor tissue. Cancer Res. 46:5786–5794; 1986.PubMedGoogle Scholar
  15. 15.
    Hammond, S. L.; Ham, R. L.; Stampfer, M. R. Serum-free growth of human mammary epithelial cells: Rapid clonal growth in defined medium and extended serial passage with pituitary extract. Cell Biol. 81:5435–5439; 1984.Google Scholar
  16. 16.
    Kotanska, K. M.; Holm, R.; Ottestad, L., et al. An ultrastructural study of benign and malignant breast epithelial cells. A search for tonofilaments. J. Submicrosc. Cytol. Pathol. 21:469–474; 1989.PubMedGoogle Scholar
  17. 17.
    Longman, S. M.; Buehring, G. C. Oral contraceptives and breast cancer. In vitro effect of contraceptive steroids on human mammary cell growth. Cancer 59:281–287; 1987.PubMedCrossRefGoogle Scholar
  18. 18.
    Malet, C.; Gompel, A.; Spritzer, P., et al. Tamoxifen and hydroxytamoxifen isomer versus extradiol effects on normal human breast cells in culture. Cancer Res. 48:7193–7199; 1988.PubMedGoogle Scholar
  19. 19.
    Maniatis, T.; Fritsch, E. F.; Sambrook, J. Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.; 1982.Google Scholar
  20. 20.
    Muller, D.; Fricker, J.; Millon-Collard, R.; et al. Characterization of cell types in human breast tumor primary cultures. Biol. Cell 61:91–99; 1987.PubMedGoogle Scholar
  21. 21.
    O'Guin, W. M.; Schermer, A.; Sum, T-T. Immunofluorescence staining of keratin filaments in cultured epithelial cells. J. Tissue Culture Methods 9(2):123–128; 1985.CrossRefGoogle Scholar
  22. 22.
    Petersen, O. W.; van Deurs, B. Preservation of defined phenotypic traits in short-term cultured human breast carcinoma derived epithelial cells. Cancer Res. 47:856–866; 1987.PubMedGoogle Scholar
  23. 23.
    Pucci-Minafra, I.; Minafra, S.; Alessandroa, R., et al. An ultrastructural evaluation of cell heterogeneity in invasive ductal carcinomas of the human breast. II. an in vitro study. J. Submicrosc. Cytol. Pathol. 21:489–499; 1989.PubMedGoogle Scholar
  24. 24.
    Pucci-Minafra, I.; Minafra, S.; Faccini, A. M., et al. An ultrastructural evaluation of cell heterogeneity in invasive ductal carcinomas of the human breast. I. an in vivo study. J. Submicrosc. Cytol. Pathol. 21:475–488; 1989.PubMedGoogle Scholar
  25. 25.
    Rudland, P. S.; Hughes, C. M.; Ferns, S. A., et al. Characterization of human mammary cell types in primary culture: Immunofluorescent and immunocytochemical indicators of cellular heterogeneity. In Vitro Cell. Dev. Biol. 25:23–36; 1989.PubMedGoogle Scholar
  26. 26.
    Russo, J.; Mills, M. J.; Moussalli, M. J., et al. Influence of human breast development on the growth properties of primary cultures. In Vitro Cell. Dev. Biol. 25:643–649; 1989.PubMedGoogle Scholar
  27. 27.
    Salhany, K. E.; Page, D. L. Fine needle aspiration of mammary lobular carcinoma in situ and atypical lobular hyperplasia. Am. J. Clin. Pathol. 92:22–26; 1989.PubMedGoogle Scholar
  28. 28.
    Siemans, C. H.; Auersperg, N. Serial propagation of human ovarian surface epithelium in tissue culture. J. Cell Physiol. 134:347–356; 1988.CrossRefGoogle Scholar
  29. 29.
    Soule, H. D.; McGrath, C. M. A simplified method for passage and long-term growth of human mammary epithelial cells. In Vitro Cell. Dev. Biol. 22:6–12; 1986.PubMedGoogle Scholar
  30. 30.
    Stampfer, M. R.; Bartely, J. C. Human mammary epithelial cells in culture: differentiation and transformation. In: Lippman, M. E.; Dickson, R. B. Breast Cancer: Cellular and Molecular Biology. Boston: Kluwer Academic Publishing; 1988:1–23.Google Scholar
  31. 31.
    Taylor-Papadimitriou, J.; Stampfer, M.; Bartek, J.; et al. Keratin expression in human mammary epithelial cells cultured from normal and malignant tissue: relation to in vivo phenotypes and influence of medium. J. Cell Sci. 94:403–413; 1989.PubMedGoogle Scholar
  32. 32.
    Yang, J.; Balakrishnan, A.; Hamamoto, S., et al. Different mitogenic and phenotypic responses of human breast epithelial cells grown in two versus three dimensions. Exp. Cell Res. 167:563–569; 1986.PubMedCrossRefGoogle Scholar
  33. 33.
    Yang, J.; Balakrishnan, A.; Hamamoto, S., et al. Human breast epithelial cells in serum-free collagen gel primary culture: growth, morphological, and immunocytochemical analysis. J. Cell. Physiol. 133:228–234; 1987.PubMedCrossRefGoogle Scholar
  34. 34.
    Zajchowski, D.; Band, V.; Pauzie, N. et al. Expression of grwoth factors and oncogenes in normal and tumor derived human mammary epithelial cells. Cancer Res. 48:7041–7047; 1988.PubMedGoogle Scholar

Copyright information

© Tissue Culture Association 1990

Authors and Affiliations

  • Joanne T. Emerman
    • 1
  • Darcy A. Wilkinson
    • 1
  1. 1.Department of AnatomyUniversity of British ColumbiaVancouverCanada

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