A method for prolonged survival of primary cell lines

Articles Cell Growth/Differentiation/Apoptosis

Summary

We have established a means for prolonged survival of primary cell cultures and establishment of continuous cell lines without genetic manipulations. Primary cultures of granulosa cells degenerate rapidly in vitro by a spontaneous onset of apoptotic cell death. Earlier attempts to circumvent this limitation have included transformation with oncogenes, spontaneous immortalization of primary cultures, and chemical carcinogenesis. We have found that addition of a complex of growth-promoting compounds, carrier proteins, and factors isolated from porcine follicular fluid to standard culture medium allows, reproducibly, the establishment of continuous porcine primary granulosa cell lines with genetic stability. This same supplement allows the prolonged survival of primary cell cultures derived from adult rat ovaries. The rat ovary primary cultures consisted of mixed phenotypes, including epithelial, neuron-like, and mesenchymal cell types. Numerous cells stain positive for alkaline phosphatase in these cultures. Other primary cell lines were established from embryonic rat liver and from adult rat lungs, using the same supplement. The survival effect is reversible because cells degenerate when the supplement is removed. Therefore, the cell lines have neither acquired properties of a tumor cell line nor have they been immortalized by a virus infection. We expect that our approach will open the door to prolonged survival of other primary cell types.

Key words

primary cell lines follicular fluid granulosa cells cell proliferation 

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References

  1. Andersen, M. M.; Krøll, J.; Byskov, A. G.; Faber, M. Protein composition in the fluid of individual bovine follicles. J. Reprod. Fertil. 48:109–118; 1976.PubMedCrossRefGoogle Scholar
  2. Bukovsky, A.; Svetlikova, M.; Caudle, M. R. Oogenesis in cultures derived from adult human ovaries. Reprod. Biol. Endocrinol. 3:17–29; 2005.PubMedCrossRefGoogle Scholar
  3. Chang, S. C. S.; Jones, J. D.; Ellefson, R. D.; Ryan, R. J. The porcine ovarian follicle: I. Selected chemical analysis of follicular fluid at different developmental stages. Biol. Reprod. 15:321–328; 1976.PubMedCrossRefGoogle Scholar
  4. Chedrese, P. J.; Rodway, M. R.; Swan, C. L.; Gillio-Meina, C. Establishment of a stable steroidogenic porcine granulosa cell line. J. Mol. Endocrinol. 20:287–292; 1988.CrossRefGoogle Scholar
  5. Donovan, P. J.; Gearhart, J. The end and the beginning for pluripotent stem cells. Nature 414:92–97; 2001.PubMedCrossRefGoogle Scholar
  6. Horisberger, M. A. Establishment of reversibly immortalized primary granulosa cell lines. Proceedings of the 37th Annual Meeting of the Union of the Swiss Societies for Experimental Research [abstract P019]; 2005.Google Scholar
  7. Hu, C.-L.; Cowan, R. G.; Harman, R. M.; Porter, D. A.; Quirk, S. M. Apoptosis of bovine granulosa cells after serum withdrawal is mediated by FAS antigen (CD95) and FAS ligand. Biol. Reprod. 64:518–526; 2001.PubMedCrossRefGoogle Scholar
  8. Joseph, N. M.; Morrison, S. J. Toward an understanding of the physiological function of mammalian stem cells. Develop. Cell 9:173–183; 2005.CrossRefGoogle Scholar
  9. Larsen, W. J.; Wert, S. E.; Chen, L.; Russell, P.; Hendrix, E. M. Expansion of the cumulus-oocyte complex during the preovulatory period: possible roles in oocyte maturation, ovulation and fertilization. In: Familiari, G.; Makabe, S.; Motta P. M., ed. Ultrastructure of the ovary. Boston: Kluwer Academic; 1991:45–61.Google Scholar
  10. Loo, D. T.; Fuquay, J. I.; Rawson, C. L.; Barnes, D. W. Extended culture of mouse embryo cells without senescence: inhibition by serum. Science 236:200–202; 1987.PubMedCrossRefGoogle Scholar
  11. Matsui, Y.; Toksoz, D.; Nishikawa, S.; Nishikawa, S.; Williams, D.; Zsebo, K.; Hogan, B. L. M. Effect of Steel factor and leukemia inhibitory factor on murine primordial germ cells in culture. Nature 353:750–752; 1991.PubMedCrossRefGoogle Scholar
  12. Matzuk, M. M.; Burns, K. H.; Viveiros, M. M.; Eppig, J. J. Intercellular communication in the mammalian ovary: oocytes carry the conversation. Science 296:2178–2180; 2002.PubMedCrossRefGoogle Scholar
  13. Motta, P. M.; Takeva, Z.; Nesci, E. Ultrastructural and histochemical study of junctions between follicular cells and the oocyte during development of ovarian follicle [in French]. Acta Anat. 80:537–562; 1971.PubMedCrossRefGoogle Scholar
  14. Motta, P. M.; Nottola, S. A.; Familiari, G.; Makabe, S.; Stallone, T.; Macchiarelli, G. Morphodynamics of the follicular-luteal complex during early ovarian development and reproductive life. Int. Rev. Cytology 223:178–288; 2003.Google Scholar
  15. Oliver, R. H.; Chen, G. D.; Yeh, J. Follicular atresia. In: Knobil, E.; Neil, J. D., ed. Encyclopedia of reproduction. Vol. 2. London: Academic Press; 1999:373–375.Google Scholar
  16. Payer, A. F. Permeability of ovarian follicles and capillaries in mice. Am. J. Anat. 142:295–318; 1975.PubMedCrossRefGoogle Scholar
  17. Polejaeva, I. A.; Chen, S. H.; Vaught, T. D., et al. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407:86–90; 2000.PubMedCrossRefGoogle Scholar
  18. Rodgers, H. F.; Lavranos, T. C.; Vella, C. A.; Rodgers, R. J. Basal lamina and other extracellular matrix produced by bovine granulosa cells in anchorage-independent culture. Cell Tissue Res. 282:463–471; 1995.PubMedGoogle Scholar
  19. Rubin, H. The disparity between human cell senescence in vitro and lifelong replication in vivo. Nature Biotechnol. 20:675–681; 2002.CrossRefGoogle Scholar
  20. Shim, H.; Gutiérrez-Adan, A.; Chen, L-R.; BonDurant, R. H.; Behhoodi, E.; Anderson, G. B. Isolation of pluripotent stem cells from cultured porcine primordial germ cells. Biol. Reprod. 57:1089–1095; 1997.PubMedCrossRefGoogle Scholar
  21. Stojkovic, M.; Lako, M.; Strachan, T.; Murdoch, A. Derivation, growth and applications of human embryonic stem cells. Reproduction 128:259–267; 2004.PubMedCrossRefGoogle Scholar
  22. Tilly, J. L.; Billig, H.; Kowalski, K. I.; Hsueh, A. J. Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism. Mol. Endocrinol. 6:1942–1950; 1992.PubMedCrossRefGoogle Scholar
  23. Yanagishita, M.; Rodbard, D.; Hascall, V. C. Isolation and characterization of proteoglycans from porcine ovarian follicular fluid. J. Biol. Chem. 254:911–920; 1979.PubMedGoogle Scholar

Copyright information

© Society for In Vitro Biology 2006

Authors and Affiliations

  1. 1.Aivogen AGBaselSwitzerland

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