In Vitro

, Volume 15, Issue 12, pp 941–948 | Cite as

Requirements for maintaining the embryonic state of avian tendon cells in culture

  • Richard I. Schwarz
  • Deborah A. Farson
  • Mina J. Bissell
Article

Summary

Primary avian tendon cells (PAT) maintain their embryonic state when cultured in medium F-12 with very low serum (0.2%) and ascorbate (50 μg per ml); that is, they retain the potential for devoting 20–30% of their total protein synthesis to collagen. However, if the cells are left at a confluent cell density or are derived from confluent cultures, this potential is irreversibly decreased. This effect, along with poor medium formulations, probably accounts for the “dedifferentiation” process that occurs when fibroblasts are cultured. In contrast, PAT cells kept at subconfluent cell densities retain the ability to synthesize high levels of collagen. The one limitation in obtaining long-term cultures of high collagen-producing tendon cells in the inability of serum at low concentrations to remain a potent mitogen after a few subcultures.

The quantitative loss of function has long been considered to be a cell culture artifact; however, we propose that this drop in collagen synthesis is a reflection of the developmental programing of these cells. In separate series of experiments using organ cultures, we show that tendon tissue from the embryo makes over 30% collagen, whereas, “young” tendons make 18% and “older” tendons from the adult make less than 1%. Therefore, a quantitative drop in collagen synthesis would be expected if normal development were to occur in culture. Our data are consistent with the idea that cultures of embryonic tendon cells are triggered to mature by a mechanism that correlates with high cell density.

Key words

collagen ascorbic acid differentiation development aging 

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References

  1. 1.
    Peterkofsky, B. 1972. Regulation of collagen secretion by ascorbic acid in 3T3 and chick embryo fibroblasts. Biochem. Biophys. Res. Commun. 49: 1343–1350.PubMedCrossRefGoogle Scholar
  2. 2.
    Peterkofsky, B., and W. B. Prather. 1974. Increased collagen synthesis in Kirsten sarcoma virus-transformed BALB 3T3 cells grown in the presence of dibutyryl cyclic AMP. Cell 3: 291–299.PubMedCrossRefGoogle Scholar
  3. 3.
    Schwarz, R., L. Colarusso and P. Doty. 1976. Maintenance of differentiation in primary cultures of avian tendon cells. Exp. Cell Res. 102: 63–71.PubMedCrossRefGoogle Scholar
  4. 4.
    Peterkofsky, B. 1972. The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured fibroblasts. Arch. Biochem. Biophys. 152: 318–328.PubMedCrossRefGoogle Scholar
  5. 5.
    Davidson, E. H. 1964. Differentiation in monolayer tissue culture cells. Adv. Genet. 12: 143–280.PubMedCrossRefGoogle Scholar
  6. 6.
    Schwarz R. I., and M. J. Bissell 1977 Dependence of the differentiated state on the cellular environment: modulation of collagen synthesis in tendon cell. Proc. Natl. Acad. Sci. U.S.A. 74: 4453–4457.PubMedCrossRefGoogle Scholar
  7. 7.
    Dehm, P., and D. J. Prockop. 1971. Synthesis and extrusion of collagen by freshly isolated cells from chick embryo tendon. Biochem. Biophys. Acta 240: 358–369.Google Scholar
  8. 8.
    Ham, R. G. 1965. Clonal growth of mammalian cells in a chemically defined, synthetic medium. Proc. Natl. Acad. Sci. U.S.A. 53: 288–293.PubMedCrossRefGoogle Scholar
  9. 9.
    Peterkofsky, B., and R. Diegelmann. 1971. Use of mixture of protease-free collagenase for specific assay of radioactive collagen in the presence of other proteins. Biochem. 10: 988–994.CrossRefGoogle Scholar
  10. 10.
    Bissell, M. J., R. C. White C. Hatié, and J. A. Bassham. 1973. Dynamics of metabolism of normal and virus-transformed chick cells in culture. Proc. Natl. Acad. Sci. U.S.A. 70: 2951–2955.PubMedCrossRefGoogle Scholar
  11. 11.
    Todaro, G. T., and H. Green. 1963. Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J. Cell. Biol. 17: 299–313.PubMedCrossRefGoogle Scholar
  12. 12.
    Diegelmann, R. F., and B. Peterkofsky. 1972. Collagen biosynthesis during connective tissue development in chick embryo. Dev. Biol. 28: 443–453.PubMedCrossRefGoogle Scholar
  13. 13.
    Peterkofsky, B., and W. Prather. 1977. Cytotoxicity of ascorbate and other reducing agents towards cultured fibroblasts as a result of hydrogen peroxide formation. J. Cell. Physiol. 90: 61–70.PubMedCrossRefGoogle Scholar
  14. 14.
    Shier, W. T., and J. T. Trotter. 1978. Oncogenic transformation of 3T3 cells associated with conversion from an “adult” to an “embryonic” esterase isoenzyme pattern. Exp. Cell Res. 111:285–294.PubMedCrossRefGoogle Scholar
  15. 15.
    Cardenas, J. M., E. Bandman, C. Walker, and R. C. Strohman. 1979. Pyruvate kinase isozymic shifts of differentiating chick myogenic cells in vivo and in culture. Dev. Biol. 68: 326–333.PubMedCrossRefGoogle Scholar
  16. 16.
    Smith, M. J. 1966. Theories of aging. In: P. L. Krohn (Ed.),Topics in the Biology of Aging. Interscience Publishers, N. Y., pp. 1–35.Google Scholar
  17. 17.
    Williams G. C. 1957. Pleiotrophy, natural selection, and the evolution of senescence. Evolution 11: 398–411.CrossRefGoogle Scholar
  18. 18.
    Bidder, G. P. 1932. Senescence. Brit. Med. J. ii: 583–585.CrossRefGoogle Scholar
  19. 19.
    Comfort, A. 1964.Ageing: The Biology of Senescence. Holt, Reinhart and Winston, Inc., N. Y.Google Scholar
  20. 20.
    Gospodarowicz, D. 1975. Purification of a fibroblast growth factor from bovine pituitary. J. Biol. Chem. 250: 2515–2520.PubMedGoogle Scholar
  21. 21.
    Rheinwald, J. G., and H. Green. 1977. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 265: 421–424.PubMedCrossRefGoogle Scholar
  22. 22.
    Cahn, R. D., and M. B. Cahn. 1966. heritability of cellular differentiation: Clonal growth and expression of differentiation in retinal pigment cells in vitro. Proc. Natl. Acad. Sci. U.S.A. 55: 106–114.PubMedCrossRefGoogle Scholar
  23. 23.
    Bornstein, P., K. von de Mark, A. W. Wyke, A. P. Erlich, and J. M. Monson. 1975. Characterization of the pro-αl chain of procollagen. J. Biol. Chem. 247: 2808–2813.Google Scholar

Copyright information

© Tissue Culture Association 1979

Authors and Affiliations

  • Richard I. Schwarz
    • 1
  • Deborah A. Farson
    • 1
  • Mina J. Bissell
    • 1
  1. 1.Laboratory of Chemical Biodynamics, Lawrence Berkeley LaboratoryUniversity of CaliforniaBerkeley

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