Establishment and characterization of a caprine mammary epithelial cell line (CMEC)

  • A. G. Pantschenko
  • J. Woodcock-Mitchell
  • S. L. Bushmich
  • T. J. Yang
Cellular Models


We describe the establishment of a continuous, nontransformed cell line obtained from primary culture of a lactating (114 days postparturition) Anglo-Nubian (Capra hircus) goat mammary gland biopsy. These cells (CMEC), have been cultured in the presence of supraphysiologic concentrations of insulin and hydrocortisone for more than 560 population doublings (over 80 passages) without any sign of senescence while maintaining a normal/near-normal diploid chromosome modal number of 2n=60 and are responsive to contact inhibition of proliferation. Cytoskeletal analysis indicates that CMECs are epithelial, without detectable fibroblastic or myoepithelial cells. When grown at low density on plastic substratum, the cells tend to form island monolayer aggregates with the characteristics cobblestone morphology of epithelial cells. With increasing density, the cells organize into lumen-like structures with various morphology consisting of large and small vacuolized and nonvacuolized cells. Postconfluent cultures form epithelial raised dome-like structures, implying a process of contact-induced differentiation. This is corroborated by positive immunocytochemistry to lactation-specific proteins: β-casein and α-lactalbumin, which were predominantly expressed in dome-forming cells. We also observed an overall modulation of cytokeratin 18/19 expression associated with number of days post subculture and with the expression of lactation-specific proteins. Postconfluent cultures which contain lactation-specific, antibody-reactive, dome-like structures showed a decreased expression of keratin 18 and no (null) expression for keratin 19. Lastly, cells cultured within a collagen matrix show morphological differentiation with the organization of branching duct-like and acini-like structures. This study suggests that CMECs are a useful in vitro model for study of mammary gland development and differentiation, in particular, direct modulation of epithelial cells grown on plastic substratum or extracellular matrix without the influence of stromal elements or the necessity and variability associated with primary cell culture or tissue explants.

Key words

differentiation dome-like structures lactogenesis cytoskeleton 


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  1. Barcellos-Hoff, M. H.; Aggeler, J.; Ram, T. G.; Bissell, M. J. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development 105:223–235; 1989.PubMedGoogle Scholar
  2. Blum, J. L.; Wicha, M. S. Role of cytoskeleton in laminin induced mammary gene expression. J. Cell. Physiol. 135:13–22; 1988.PubMedCrossRefGoogle Scholar
  3. Cifrian, E.; Guidry, A. J.; O'Brien, C..; Nickerson, S. C.; Marquardt, W. W. Adherence of Staphylococcus aureus to cultured bovine mammary epithelial cells. J. Dairy Sci. 77:970–983; 1994.PubMedGoogle Scholar
  4. Dairkee, S. H.; Blayney, C. M.; Asarnow, D. M.; Smith, H. S.; Hackett, A. J. Early expression of vimentin in human mammary cultures. In Vitro Cell. Dev. Biol. 21:321–327; 1985.PubMedGoogle Scholar
  5. Danielson, K. G.; Osborn, C. J.; Durban, E. M.; Butel, J. S.; Medina, D. Epithelial mouse mammary cell line exhibiting normal morphogenesis in vivo and functional differentiation in vitro. Proc. Natl. Acad. Sci. USA 81:3756–3760; 1984.PubMedCrossRefGoogle Scholar
  6. Dils, R.; Forsyth, I. A. Preparation and cultures of mammary gland explants. In Methods in enzymology, Vol. 72. Cell and tissue techniques. New York: Academic Press; 1981:732–733.Google Scholar
  7. Ethier, S. P. Human breast cancer cell lines as models of growth regulation and disease progression. J. Mammary Gland Biol. Neoplasia 1:111–121; 1996.PubMedCrossRefGoogle Scholar
  8. Forsyth, I. A.; Turvey, A. Fatty acid synthesis by explant cultures from the mammary glands of goats on days 60 and 120 of pregnancy. J. Endocrinol 100:87–92; 1984.PubMedCrossRefGoogle Scholar
  9. Freshney, R. I. Contamination. In: Culture of animal cells: a manual of basic technique. 2nd ed. New York: Wiley-Liss; 1990:208–211.Google Scholar
  10. Goff, S; Traktman, P.; Baltimore, D. Isolation and properties of moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase. J. Virol. 38:239–248; 1981.PubMedGoogle Scholar
  11. Hancock K.; Tsang, V. C. M.. India ink staining of proteins on nitrocellulose paper. Anal. Biochem. 133:157–162; 1983.PubMedCrossRefGoogle Scholar
  12. Howlett, A. R.; Bissell, M. J., Regulation of mammary epithelial cell function: a role for stromal and basement membrane matrices. Protoplasma 159:85–95; 1990.CrossRefGoogle Scholar
  13. Hurley, W. L., Mammary gland function during involution. J. Dairy Sci., 72: 1637–1646; 1989.PubMedGoogle Scholar
  14. Huynh, H. T.; Robitaille, G.; Turner, J.. Establishment of bovine mammary epithelial cells (MAC-T): an in vitro model for bovine lactation. Exp. Cell Res. 197:191–199; 1991.PubMedCrossRefGoogle Scholar
  15. Huynh, H. T.; Pollak, M.. HH2a, an immortalized bovine mammary epithelial cell line expreses the gene encoding mammary derived growth inhibitor (MDGI). In Vitro Cell Dev. Biol. 31A:25–29; 1995.Google Scholar
  16. Ilan, N.; Barash, I. Gootwine, E.; Shani, M. Establishment and initial characterization of the ovine mammary epithelial cell line NISH. In Vitro Cell. Dev. Biol. 34:326–332; 1998.Google Scholar
  17. Ip, M. M.; Darcy, K. M. Three-dimensional mammary primary culture model systems. J. Mammary Gland Biol. Neoplasia 1:91–110; 1996.PubMedCrossRefGoogle Scholar
  18. Kleinman, H. K.; McGarvey, M. I.; Hassell, J. R.; Star, V. L.; Cannon, F. B.; Laurie, G. W.; Martin, G. R. Basement membrane complexes with biological activity. Biochemistry 25:312–318; 1986.PubMedCrossRefGoogle Scholar
  19. Knight, C. H.; Peaker, M. Mammary development and regression during lactation in goats in relation to milk secretion. Quart. J. Exp. Physiol. 69:331–338; 1984.Google Scholar
  20. Knight, C. H. Hillerton, J. E.; Teverson, R. A.; Winter, A. Biopsy of the bovine mammary gland. Br. Vet. J. 148:129–132; 1992.PubMedGoogle Scholar
  21. Knight, C. H.; Widle, C. J. Mammary growth during lactation: implications for increasing milk yield. J. Dairy Sci. 70:1991–2000; 1987.PubMedGoogle Scholar
  22. Koch, F. C. Preparation of casein from skimmed milk. In: Practical methods in biochemistry. 2nd ed. Baltimore: William Wood & Co.; 1937:58–59.Google Scholar
  23. Laemmli, U. K. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227:680–685; 1970.CrossRefGoogle Scholar
  24. Li, P.; Fernig, D. G.; Rudland, P. S.; Fich, L. M.; Wilde, C. Identification of cell types in the developing goat mammary gland. Biochem. Soc. Trans. 24:357S; 1996.PubMedGoogle Scholar
  25. MacPherson, I. Soft agar techniques. In: Kruse, P. F.; Patterson, M. K., Jr., ed. Methods in enzymology. Vol. XXVII. Tissue culture methods and applications. New York: Academic Press; 1973:276–280.Google Scholar
  26. Mensher, S. H.; Bunch, T. D.; Maciulis, A. High-resolution G-banding karyo-type and idiogram of the goat: a sheep-goat G-banded comparison. J. Heredity. 80:150–155; 1989.Google Scholar
  27. Michalopoulos, G.; Pilot, H. C. Primary culture of parenchymal liver cells on collagen membranes: morphological and biochemical observations. Exp. Cell Res. 94:70–78; 1975.PubMedCrossRefGoogle Scholar
  28. Mitchell, J. J.; Woodcock-Mitchell, J.; Reynolds, S.; Low, R. B.; Leslic, K.; Adler, K.; Gabiani, G.; Skalli, O. α-smooth muscle actin in parenchymal cells of bleomycin injured rat lung. Lab. Invest. 60:643–650; 1989.PubMedGoogle Scholar
  29. Mitchell, J. J.; Low, R. B.; Woodcock-Mitchell, J. Cytomatrix synthesis in MDCK epithelial cells. J. Cell. Physiol. 143:501–511; 1990.PubMedCrossRefGoogle Scholar
  30. Oliver, S. P.; Sordillo, L. M. Approaches to the manipulation of mammary involution. J. Dairy Sci. 72:1647–1664; 1989.PubMedGoogle Scholar
  31. Parry, G.; Lee, E.-H.; Farso, D. A.; Kovall, N.; Bissell, M. J. Collagenous substrata regulated the nature and distribution of glycosaminoglycans produced by differentiated cultures of mouse mammary epithelial cell. Exp. Cell Res. 156:487–499; 1985.PubMedCrossRefGoogle Scholar
  32. Parry, G.; Cullen, R.; Kaetzel, C. S.; Kramer, R.; Moss, L. Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences. J. Cell Biol. 105:2043–2051; 1987.PubMedCrossRefGoogle Scholar
  33. Pickett, P. B.; Pitelka, D. R.; Hamamoto, S. T.; Misfeldt, D. S. Occluding junctions and cell behavior in primary cultures of normal and neoplastic mammary gland cells. J. Cell Biol. 66:316–332; 1975.PubMedCrossRefGoogle Scholar
  34. Sapino, A.; Macri, L.; Gugliotta, P.; Bussolati, G. Immunocytochemical identification of proliferating cell types in the mouse mammary gland. J. Histochem. Cytochem. 38:1541–1547; 1990.PubMedGoogle Scholar
  35. Schalm, O. W.; Carroll, E. J.; Jain, N. C. Physical and chemical tests for detection of mastitis. In: Bovine mastitis. Philadelphia: Lea & Febiger; 1971:150–151.Google Scholar
  36. Schmid, E.; Schiller, D. L.; Grund, C.; Stadler, J.; Franke, W. W. Tissue type-expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland. J. Cell Biol. 96:37–50; 1983a.PubMedCrossRefGoogle Scholar
  37. Schmid, E.; Franke, W. W.; Grund, C.; Schiller, D. L.; Kolb, H.; Paweletz, N. An epithelial cell line with elongated myod morphology derived from bovine mammary gland: expression of cytokeratins and desmosomal plaque proteins in unusual arrays. Exp. Cell Res. 146:309–328; 1983b.PubMedCrossRefGoogle Scholar
  38. Shay, J. W.; Pereira-Smith, O. M.; Wright, W. E. A role for both RB and p53 in the regulation of human cellular senescence. Exp. Cell Res. 196: 33–39; 1991.PubMedCrossRefGoogle Scholar
  39. Skalli, O.; Ropraz, P.; Trzeciak, A.; Benzonana, G.; Gillessen, D.; Gabbiani, G. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J. Cell Biol. 103:2787–2796; 1986.PubMedCrossRefGoogle Scholar
  40. Stampfer, M.; Hallowers, R. C.; Hackett, A. J. Growth of normal human mammary cell in culture. In Vitro Cell. Dev. Biol. 16:415–425; 1980.CrossRefGoogle Scholar
  41. Streuli, C. H.; Bissell, M. J. Expression of extracellular matrix components is regulated by substratum. J. Cell Biol. 10:1405–1415; 1990.CrossRefGoogle Scholar
  42. Taylor-Papadimitriou, J.; Stampfer, M.; Bartek, J.; Lewis, A.; Boshell, M.; Lane, E. B.; Leigh, I. M. Keratin expression in human mammary epithelial cells cultures from normal and malignant tissue: relation to in vivo phenotypes and influence of medium. J. Cell Sci. 94:403–413; 1989.PubMedGoogle Scholar
  43. Towbin, H.; Staehelin, T.; Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc. Natl. Acad. Sci. USA 76:4350–4354; 1979.PubMedCrossRefGoogle Scholar
  44. Warburton, M. J.; Head, L. P.; Rudland, P. S. Redistribution of fibronectin and cytoskeletal proteins during the differentiation of rat mammary tumor cell in vitro. Exp. Cell Res. 132:57–66; 1981.PubMedCrossRefGoogle Scholar
  45. Wilde, C. J.; Knight, C. H. Metabolic adaptations in mammary gland during the declining phase of lactation. J. Dairy Sci. 72:1679–1692; 1989.PubMedCrossRefGoogle Scholar
  46. Willey, R. L.; Smith, D. H.; Lasky, L. A.; Theodore, T. S.; Earl, P. L.; Moss, B.; Capon, D. J.; Nartin, M. A. In vitro mutagenesis identifies a region within the envelope gene of human immunodeficiency virus that is critical for infectivity. J. Virol. 2:139–147; 1988.Google Scholar
  47. Woessner, J. F., Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid. Arch. Biochem. Biophysics. 93:440–447; 1961.CrossRefGoogle Scholar
  48. Worton, R. G.; Duff, C. Karyotyping. In: Jakoby, W. B.; Pastan, I. H., ed. Methods in enzymology. Vol. LVIII. Cell culture. New York: Academic Press; 1979:322–344.Google Scholar
  49. Yang, T. J. Assay for colony formation in semisolid agar of cells treated with agents with agglutinating property. TCA (Tissue Cult. Assoc.) Manual 4:845–848; 1978.CrossRefGoogle Scholar
  50. Zavizion, B.; Politis, I.; Gorewit, R. C. Bovine mammary myoepithelial cells. 2. interactions with epithelial cells in vitro. J. Dairy Sci. 75:3381–3393; 1992.PubMedCrossRefGoogle Scholar
  51. Zavizion, B.; VanDuffelen, M.; Schaeffer, W.; Politis, I. Use of microinjection to generate an immortalized bovine mammary cell line with both epithelial and myoepithelial characteristics. Methods Cell Sci. 17:271–282; 1995.CrossRefGoogle Scholar
  52. Zavizion, B.; VanDuffelen, M.; Schaeffer, W.; Politis, I. Establishment and characterization of a bovine mammary epithelial cell line with unique properties. In Vitro Cell. Dev. Biol. 32:138–148; 1996.Google Scholar

Copyright information

© Society for In Vitro Biology 2000

Authors and Affiliations

  • A. G. Pantschenko
    • 1
  • J. Woodcock-Mitchell
    • 2
  • S. L. Bushmich
    • 1
  • T. J. Yang
    • 1
  1. 1.Department of PathobiologyUniversity of ConnecticutStorrs
  2. 2.Department of Molecular Physiology and Biophysics, College of MedicineUniversity of VermontBurlington

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