Cytotechnology

, Volume 55, Issue 1, pp 9–13 | Cite as

Establishment of primary cell cultures from fish calcified tissues

  • Cátia L. Marques
  • Marta S. Rafael
  • M. Leonor Cancela
  • Vincent Laizé
Technical Note

Abstract

Fishes have been recently recognized as a suitable model organism to study vertebrate physiological processes, in particular skeletal development and tissue mineralization. However, there is a lack of well characterized in vitro cell systems derived from fish calcified tissues. We describe here a protocol that was successfully used to develop the first calcified tissue-derived cell cultures of fish origin. Vertebra and branchial arches collected from young gilthead seabreams were fragmented then submitted to the combined action of collagenase and trypsin to efficiently release cells embedded in the collagenous extracellular matrix. Primary cultures were maintained under standard conditions and spontaneously transformed to form continuous cell lines suitable for studying mechanisms of tissue mineralization in seabream. This simple and inexpensive protocol is also applicable to other calcified tissues and species by adjusting parameters to each particular case.

Keywords

Calcified tissues Gilthead seabream (Sparus aurata L.) In vitro cell system Mineralization Cell culture protocol 

Abbreviations

DMSO

Dimethyl sulphoxide

PEI

Polyethylenimine

ECM

Extracellular matrix

References

  1. Bejar J, Borrego JJ, Alvarez MC (1997) A continuous cell line from the cultured marine fish gilt-head seabream (Sparus aurata L.). Aquaculture 150:143–153CrossRefGoogle Scholar
  2. Berghmans S, Jette C, Langenau D, Hsu K, Stewart R, Look T, Kanki JP (2005) Making waves in cancer research: new models in the zebrafish. Biotechniques 39:227–237CrossRefGoogle Scholar
  3. Bolis CL, Piccolella M, Dalla Valle AZ et al (2001) Fish as model in pharmacological and biological research. Pharmacol Res 44:265–280CrossRefGoogle Scholar
  4. Braga D, Laizé V, Tiago DM, Cancela ML (2006) Enhanced DNA transfer into fish bone cells using polyethylenimine. Mol Biotechnol 34:51–54CrossRefGoogle Scholar
  5. Costa MA, Fernandes MH (2000) Long-term effects of parathyroid hormone, 1,25-dihydroxyvitamin d(3), and dexamethasone on the cell growth and functional activity of human osteogenic alveolar bone cell cultures. Pharmacol Res 42:345–353CrossRefGoogle Scholar
  6. Crane MS (1999) Mutagensis and cell transformation in cell culture. Methods Cell Sci 21:245–253CrossRefGoogle Scholar
  7. Fernandez RD, Yoshimizu M, Kimura T, Ezura Y (1993) Establishment and characterization of seven continuous cell lines from freshwater fish. J Aquat Anim Health 5:137–147CrossRefGoogle Scholar
  8. Fisher S, Jagadeeswaran P, Halpern ME (2003) Radiographic analysis of zebrafish skeletal defects. Dev Biol 264:64–76CrossRefGoogle Scholar
  9. Fonseca VG, Laizé V, Valente MS, Cancela ML (2007) Identification of an osteopontin-like protein in fish associated with mineral formation. FEBS J 274:4428–4439CrossRefGoogle Scholar
  10. Fournier B, Price PA (1991) Characterization of a new human osteosarcoma cell line OHS-4. J Cell Biol 114:577–583CrossRefGoogle Scholar
  11. Hightower LE, Renfro JL (1988) Recent applications of fish cell culture to biomedical research. J Exp Zool 248:290–302CrossRefGoogle Scholar
  12. Kellermann O, Buc-Caron MH, Marie PJ, Lamblin D, Jacob F (1990) An immortalized osteogenic cell line derived from mouse teratocarcinoma is able to mineralize in vivo and in vitro. J Cell Biol 110:123–132CrossRefGoogle Scholar
  13. Kelly KA, Havrilla CM, Brady TC, Abramo KH, Levin ED (1998) Oxidative stress in toxicology: established mammalian and emerging piscine model systems. Environ Health Perspect 106:375–384CrossRefGoogle Scholar
  14. Laizé V, Pombinho AR, Cancela ML (2005) Characterization of Sparus aurata osteonectin cDNA and in silico analysis of protein conserved features: evidence for more than one osteonectin in Salmonidae. Biochimie 87:411–420CrossRefGoogle Scholar
  15. McGonnell IM, Fowkes RC (2006) Fishing for gene function—endocrine modelling in the zebrafish. J Endocrinol 189:425–439CrossRefGoogle Scholar
  16. Nissen RM, Amsterdam A, Hopkins N (2006) A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression. BMC Dev Biol 6:28CrossRefGoogle Scholar
  17. Patton EE, Zon LI (2001) The art and design of genetic screens: zebrafish. Nat Rev Genet 2:956–966CrossRefGoogle Scholar
  18. Pombinho AR, Laizé V, Molha DM, Marques SM, Cancela ML (2004) Development of two bone-derived cell lines from the marine teleost Sparus aurata; Evidence for extracellular matrix mineralization and cell-type-specific expression of matrix Gla protein and osteocalcin. Cell Tissue Res 315:393–406CrossRefGoogle Scholar
  19. Rafael MS, Laizé V, Cancela ML (2006) Identification of Sparus aurata bone morphogenetic protein 2: molecular cloning, gene expression and in silico analysis of protein conserved features in vertebrates. Bone 39:1373–1381CrossRefGoogle Scholar
  20. Stanford CM, Jacobson PA, Eanes ED, Lembke LA, Midura RJ (1995) Rapidly forming apatitic mineral in an osteoblastic cell line. J Biol Chem 270:9420–9428CrossRefGoogle Scholar
  21. Wolf K, Mann JA (1980) Poikilotherm vertebrate cell lines and viruses: a current listing for fishes. In Vitro 16:168–179Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Cátia L. Marques
    • 1
  • Marta S. Rafael
    • 1
  • M. Leonor Cancela
    • 1
  • Vincent Laizé
    • 1
  1. 1.Centre of Marine Sciences (CCMAR)University of AlgarveFaroPortugal

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