Advertisement

In Vitro - Plant

, Volume 18, Issue 10, pp 817–826 | Cite as

A method for culturing chick melanocytes: The effect of BRL-3A cell conditioning and related additives

  • Barbara Giss
  • Jane Antoniou
  • Gary Smith
  • John Brumbaugh
Article

Summary

A method for growing chick embryo melanocytes is described that utilizes medium conditioned by Buffalo Rat liver (BRL-3A) cells. The dissected trunk region of each 72 h (Stages 14 to 19) embryo produces approximately 200,000 melanocytes (purity, 80%) when processed and cultured for 8 d. Thus, a typical experiment involving 20 embryos would produce a total of 4 × 106 melanocytes. Choice of serum, serum concentration, and cell density were determined experimentally. Partially purified multiplication stimulating activity (MSA) from BRL-3A cells and insulin were also tested as medium additives. MSA was not stimulatory, whereas insulin gave a positive response in 2% but not 10 or 0% serum. The final protocol used a modified F12 medium with 10% bovine calf serum conditioned by BRL-3A cells. Cultures were fed every other day. Small colonies of cells became evident by culture Day 3 and increased rapidly to Day 5 when pigmentation became obvious. Colony size continued to increase but more slowly from Days 5 to 8, whereas pigmentation increased rapidly and maximized on Day 8. There is a factor, or factors, present in BRL-3A conditioned medium that stimulates embryonic chick melanocytes to divide preferentially over contaminating cell types. This results in cultures that can provide adequate numbers and purity for biochemical studies.

Key words

chick melanocytes BRL-3A conditioning multiplication stimulating activity (MSA) insulin pigmentation 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Zimmerman, J.; Brumbaugh, J.; Biehl, J.; Holtzer, H. The effect if 5-bromodeoxyuridine on the differentiation of chick embryo pigment cells. Exp. Cell Res. 83: 159–165; 1974.PubMedCrossRefGoogle Scholar
  2. 2.
    Brumbaugh, J.; Wilkins, L.; Schall, D. Intergenic complementation in chick melanocyte heterokaryons. Exp. Cell Res. 111: 333–341; 1978.PubMedCrossRefGoogle Scholar
  3. 3.
    Cohen, A.; Konigsberg, I. A clonal approach to the problem of neural crest determination. Dev. Biol. 46: 262–280; 1975.PubMedCrossRefGoogle Scholar
  4. 4.
    Loring, J.; Glimelius, B.; Erickson, C.; Weston, J. Analysis of developmentally homogeneous neural crest cell populationsin vitro. I. Formation, morphology and differentiative behavior. Dev. Biol. 82: 86–94; 1981.PubMedCrossRefGoogle Scholar
  5. 5.
    Maxwell, D. Cell cycle changes during neural crest cell differentiationin vitro. Dev. Biol. 49: 66–79; 1976.PubMedCrossRefGoogle Scholar
  6. 6.
    Glimelius, B.; Weston, J. Analysis of developmentally homogeneous neural crest cell populationsin vitro. II. A tumor-promoter (TPA) delays differentiation and promotes cell proliferation. Dev. Biol. 82: 95–101; 1981.PubMedCrossRefGoogle Scholar
  7. 7.
    Mayer, T.; Oddis, L. Pigment cell differentiation in embryonic mouse skin and isolated epidermis: Anin vitro study. J. Exp. Zool. 202: 415–424; 1977.PubMedCrossRefGoogle Scholar
  8. 8.
    Mayer, T. The relationship between cell division and melanocyte differentiation in epidermal cultures from mouse embryos. Dev. Biol. 79: 419–427; 1980.PubMedCrossRefGoogle Scholar
  9. 9.
    Wilkins, L.; Szabo, G.; Maciag, T.; Nemore, R.; Gilchrest, B. A system forin vitro growth of human epidermal melanocytes. J. Invest. Dermatol. 76: 315; 1981.Google Scholar
  10. 10.
    Barnes, D.; Sato, G. Serum-free cell culture: A unifying approach. Cell 22: 649–655; 1980.PubMedCrossRefGoogle Scholar
  11. 11.
    Smith, G.; Temin, H. Purified multiplication-stimulating activity from rat liver cell conditioned medium: Comparison of biological activities with calf serum, insulin, and somatomedin. J. Cell Physiol. 84: 181–192; 1974.PubMedCrossRefGoogle Scholar
  12. 12.
    Dulak, N.; Temin, H. A partially purified polypeptide fraction from rat liver cell conditioned medium with multiplication-stimulating activity for embryo fibroblasts. J. Cell. Physiol. 81: 153–160; 1973.PubMedCrossRefGoogle Scholar
  13. 13.
    Dulak, N.; Temin, H. Multiplication-stimulating activity from rat liver cell conditioned medium: A family of small polypeptides. J. Cell Physiol. 81: 161–170; 1973.PubMedCrossRefGoogle Scholar
  14. 14.
    Moses, A.; Wissley, S.; Short, P.; Rechler, M.; Podskalny, J. Purification and characterization of multiplication-stimulating activity. Eur. J. Biochem. 103: 387–400; 1980.PubMedCrossRefGoogle Scholar
  15. 15.
    Rechler, M.; Nissley, S.; King, G.; Moses, A.; Van Obberghen-Schilling, E.; Romanus, J.; Knight, A.; Short, P.; White, R. Multiplication-stimulating activity (MSA) from the BRL-3A rat liver cell line: Relation to human somatomedins and insulin. J. Supramol. Struct. and Cell. Biochem. 15: 253–286; 1981.CrossRefGoogle Scholar
  16. 16.
    Brumbaugh, J.; Hollander, W. A further study of theE pattern locus in the fowl. Iowa State J. Sci. 40: 51–64; 1965.Google Scholar
  17. 17.
    Hamburger, V.; Hamilton, H. A series of normal stages in the development of the chick embryo. J. Morphol. 88: 49–92; 1951.CrossRefGoogle Scholar
  18. 18.
    McKeehan, W.; Hamilton, W.; Ham, R. Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts. Proc. Natl. Acad. Sci. USA 73: 2023–2027; 1976.PubMedCrossRefGoogle Scholar
  19. 19.
    Hayflick, L. Tissue culture and mycoplasma. Tex. Rep. Biol. Med. 23 (Suppl. I): 285–303; 1965.PubMedGoogle Scholar
  20. 20.
    Knauer, D.; Wagner, F.; Smith, G. Purification and characterization of multiplication-stimulating activity (MSA) carrier protein. J. Supramol. Struct. and Cell. Biochem. 15: 177–191; 1981.CrossRefGoogle Scholar
  21. 21.
    Paul, J. Cell and tissue culture, 5th ed. Edinburgh: Churchill Livingstone; 1975.Google Scholar
  22. 22.
    Bottenstein, J.; Sato, G.; Mather, J. Growth of neuroepithelial-derived cell lines in serum-free, hormone-supplemented media. Sato, G.; Ross, R. eds. Hormones and cell culture, Vol. 6. New York: Cold Spring Harbor Laboratory; 1979: 531–544.Google Scholar
  23. 23.
    Knauer, D.; Smith, G. Inhibition of biological activity of multiplication-stimulating activity by binding to its carrier protein. Proc. Natl. Acad. Sci. USA 77: 7252–7256; 1980.PubMedCrossRefGoogle Scholar
  24. 24.
    Snedecor, G. Statistical methods. Ames, Iowa: Iowa State College Press; 1956: 173–175.Google Scholar
  25. 25.
    Sieber-Blum, M.; Cohen, A. Clonal analysis of quail neural crest cells: They are pluripotent and differentiatein vitro in the absence of noncrest cells. Dev. Biol. 80: 96–106; 1980.PubMedCrossRefGoogle Scholar
  26. 26.
    Glimelius, B.; Weston, J. Analysis of developmentally homogeneous neural crest cell populationsin vitro. III. Role of culture environment in cluster formation and differentiation. Cell Differ. 10: 57–67; 1981.PubMedCrossRefGoogle Scholar
  27. 27.
    Brumbaugh, J.; Lee, K. The gene action and function of two dopa oxidase positive melanocyte mutants of the fowl. Genetics 81: 333–347; 1975.PubMedGoogle Scholar
  28. 28.
    Rechler, M.; Zapf, J.; Nissley, S.; Froesch, E.; Moses, A.; Podskalny, J.; Schilling, E.; Humbel, R. Interactions of insulin-like growth factors I and II and multiplication-stimulating activity with receptors and serum carrier proteins. Endocrinology 107: 1451–1459; 1980.PubMedCrossRefGoogle Scholar
  29. 29.
    Strauss, D.; Coppcock, D.; Pang, K. Low molecular weight mitogenic factor produced by BRL-3A cultured rat liver cells. Biochem. Biophys. Res. Commun. 100: 1619–1625; 1981.CrossRefGoogle Scholar

Copyright information

© Tissue Culture Assoc. Inc. 1982

Authors and Affiliations

  • Barbara Giss
    • 1
  • Jane Antoniou
    • 1
  • Gary Smith
    • 1
  • John Brumbaugh
    • 1
  1. 1.School of Life SciencesUniversity of NebraskaLincoln

Personalised recommendations