Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Growth and differentation of rat mammary epithelial cells cultured in serum-free medium

Abstract

A new serum-free defined medium was developed that supports the growth of normal rat mammary epithelial cells. Mammary organoids from the glands of female F344 rats were cultured in a serum-free medium. Monolayer culture colonies developed within a week and remained viable for months in culture. Upon subculture of one-week-old primary colonies, almost the same morphology of colonies was developed. The scrape loading/dye transfer technique showed that most of colonies that developed in a serum-free medium containing EGF, human transferrin, insulin, and hydrocortisone (basal serum-free medium, BSFM) failed to show cell-cell communication. However, colonies cultured in BSFM supplemented with prolactin, E2, and progesterone (complete hormone serum-free medium, CHSFM) showed cell-cell communication at 14 days of primary culture or of subculture. By flow cytometry with FITC-PNA and PE-anti-Thy-1.1 monoclonal antibody, we distinguished four RMEC subpopulations in cultures in both media: Thy-1.1+ cells, PNA+ cells, cells negative to both reagents and cells positive to both reagents. It is likely that combined prolactin, cortisol, and insulin in CHSFM stimulate terminal differentiation of clonogenic cells.

This is a preview of subscription content, log in to check access.

References Cited

  1. Barraclough, R., Dawson, K. J., and Rudland, P. S., Elongated cells derived from rat mammary cuboidal epithelial cell lines resemble cultured mesenchymal cells in their pattern of protein synthesis.Biochem. Biophys. Res. Commun., 120, 351–358 (1984).

  2. Barraclough, R., Kimbell, R., and Rudland, P. S., Differential control of mRNA levels for Thy-1 antigen and laminin in rat mammary epithelial and myoepithelial-like cells.J. Cell. Physiol., 131, 393–401 (1987).

  3. Chang, C. C., Trosko, J. E., El-Fouly, M. H., Gibson-D’Ambrosio, R. E., and D'Ambrosio, S. M., Contact insensitivity of a subpopulation of normal human fetal kidney epithelial cells and of human carcinoma cell lines.Cancer Res., 47, 1634–1645 (1987).

  4. Clifton, K. H., Sridharan, B. N., and Douple, E. B., Mammary carcinogenesis-enhancing effect of adrenalectomy in irradiated rats with pituitary tumor MtT-F4.J. Natl. Cancer Inst., 55, 485–487 (1975).

  5. Clifton, K. H. and Crowley, J., Effects of radiation type and role of glucocorticoids, gonadectomy and thyroidectomy in mammary tumor induction in MtT-grafted rats.Cancer Res., 38, 1507–1513 (1978).

  6. Clifton, K. H. and Gould, M. N., Clonogen transplantation assay of mammary and thyroid epithelial cells. In Potten, C. S. and Hendry, J. H. (Eds.).Cell Clones: Manual of Mammalian Cell Techniques, Churchill Livingstone, London, pp. 128–138, 1985.

  7. Clifton, K. H., The clonogenic cells of the rat mammary and thyroid glands: Their biology, frequency of initiation and promotion/progression to cancer. In Moolgavkar, S. H. (Ed.).Scientific Issues in Quantitative Cancer Risk Assessment, Birkhauser, Boston, pp. 1–21, 1990.

  8. Coleman, S., Silberstein, G. B., and Daniel, C. W., Ductal morphogenesis in the mouse mammary gland: evidence supporting a role for epidermal growth factor.Dev. Biol., 127, 304–315 (1988).

  9. Deeks, S., Richards, J., and Nandi, S., Maintenance of normal rat mammary epithelial cells by insulin and insulin-like growth factor 1.Exp. Cell Res., 174, 448–460 (1988).

  10. Dulbecco, R., Allen, W. R., Bologna, M., and Bowman, M., Marker evolution during the development of the rat mammary gland: stem cells identified by markers and the role of myoepithelial cells.Cancer Res., 46, 2449–2456 (1986).

  11. Ethier, S. P., Kudla, A., and Cundiff, K. C., Influence of hormone and growth factor interactions on the proliferative potential of normal rat mammary epithelial cellsin vitro.J. Cell. Physiol., 132, 161–167 (1987).

  12. Gould, M. N., Biel, W. F., and Clifton, K. H., Morphological and quantitative studies of gland formation from inocula of monodispersed rat mammary cells.Exp. Cell Res., 107, 405–416 (1977).

  13. Hahm, H. A. and Ip, M. M., Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. I. Regulation of proliferation by hormones and growth factors.In Vitro, 26, 792–802 (1990).

  14. Hammond, S. L., Ham, R. G., and Stampfer, M. R., Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract.Proc. Natl. Acad. Sci. USA., 81, 5435–5439 (1984).

  15. Imagawa, W., tomooka, Y., and Hamamoto, S., Stimulation of mammary epithelial cell growthin vitro: interaction of epidermal growth factor and mammogenic hormones.Endocrinology, 116, 1514–1524 (1985).

  16. Kamiya, K., Kim, N. D., Gould, M. N., and Clifton, K. H., Repair of potentially lethal damage in rat mammary clonogens following irradiation in organoid culture.Int. J. Radiat. Biol., 59, 1207–1216 (1991).

  17. Kidwell, W. R., Salomon, D. S., and Liotta, L. A., Effects of growth factors on mammary epithelial cell proliferation and basement membrane synthesis. In Barnes, D., Sato, G. (Eds.),Growth of cells in hormonally defined medium, Alan R. Liss, Inc., New York, vol. 2, pp. 807–818, 1984.

  18. Kim, N. D., and Clifton, K. H., Characterization of rat mammary epithelial cell subpopulations by peanut lectin and anti-Thy-1.1 antibody and study of flow sorted cellsin vivo.Exp. Cell Res., 207, 74–85 (1993).

  19. Kim, N. D., Oberley, T. D., and Clifton, K. H., Primary culture of flow cytometry-sorted rat mammary epithelial cell (RMEC) subpopulations in a reconstituted basement membrane, Matrigel.Exp. Cell Res., 209, 6–20 (1993).

  20. Kim, N. D., and Piak, K. J., Study of rat mammary epithelial stem cellsin vivo andin vitro.J. Korean Soc. Food Nutr., 24, 470–486 (1995).

  21. Klein, P. J., Newman, R. A., M ller, P., Uhlenbruck, G., Citoler, P., Schaefer, H. E., Lennartz, K. J., and Fischer, R., The presence and significance of the Thomsem-Friedenreich antigen in mammary gland.J. Cancer Res. Clin. Oncol., 93, 205–214 (1979).

  22. McGrath, M., Palmer, S., and Nandi, S., Differential response of normal rat mammary epithelial cells to mammogenic hormones and EGF.J. Cell. Physiol., 125, 182–191 (1985).

  23. Morris, R. J. and Ritter, M. A., Association of Thy-1 cell surface differentiation antigen with certain connective tissuesin vivo.Cell Tissue Res., 206, 459–475 (1980).

  24. Newman, R. A., Klein, P. J., and Rudland, P. S., Binding of peanut lectin to breast epithelium, human carcinomas and a cultured rat mammary stem cell and its use as a marker of mammary differentiation.J. Natl. Cancer Inst., 63, 1339–1346 (1979a).

  25. Newman, R. A., Kelin, P. J., Uhlenbruck, G., Citoler, P., and Karduck, D., The presence and significance of the Thomsen-Friedenreich antigen in breast cancer. I. Serological studies.J. Cancer Res. Clin. Oncol., 93, 181–188 (1979b).

  26. Porter, E. H., Hewitt, H. B., and Blake, E. R., The transplantation kinetics of tumour cells.Br. J. Cancer, 27, 55–62 (1973).

  27. Rudland, P. S. and Barraclough, R., Stem cells in mammary gland differentiation and cancer.J. Cell Sci. Suppl., 10, 95–114 (1988).

  28. Rudland, P. S., Generation of lobuloalveolar development from isolated rat mammary ducts and end buds.J. Histochem. Cytochem., 39, 1257–1266 (1991).

  29. Salomon, D. S., Liotta, L. A., and Kidwell, W. R., Differential response to growth factgor by rat mammary eoithelium plated on different collagen substrata in serum-free medium.Proc. Natl. Acad. Sci. USA, 78, 382–386 (1981).

  30. Trosko, J. E., and Chang, C. C., Stem cell theory of carcinogenesis.Toxicol. Lett., 49, 283–295 (1989).

  31. Vonderhaar, B. K., Local effects of EGF, TGF-, and EGF-like growth factors on lobuloalveolar development of the mouse mammary glandin vivo.J. Cell. Physiol., 132, 581–584 (1987).

  32. Warburton, M. J., Ferns, S. A., Hughes, C. M., and Rudland, P. S., Characterization of rat mammary cells types in primary culture: lectins and antisera to basement membrane and intermediate filatment proteins as indicators of cellular heterogeneity.J. Cell Sci., 79, 287–304 (1985).

  33. Welte, K., Platzer, E., Lu, L., Gabrilove, J. L., Levi, E., Mertelsmann, R., and Moore, M. A., Purification and biochemical characterization of human pluripotent hematopoietic colony-stimulating factor.Proc. Natl. Acad. Sci. USA, 82, 1526–1530 (1985).

  34. Yang, J., Larson, L., and Flynn, D., Serum-free primary culture of human normal mammary epithelial cells in collagen gel matrix.Cell Biol. Int. Rep., 6, 969–975 (1982).

Download references

Author information

Correspondence to Nam Deuk Kim.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kim, D.Y., Jhun, B., Lee, K.H. et al. Growth and differentation of rat mammary epithelial cells cultured in serum-free medium. Arch. Pharm. Res. 20, 297 (1997). https://doi.org/10.1007/BF02976190

Download citation

Key words

  • Mammary epithelial cell
  • Differentiation
  • Flow cytometry
  • Cell-cell communication