, Volume 16, Issue 2, pp 109–120 | Cite as

Identification of media supplements that improve the viability of primarily cell cultures ofCrassostrea gigas oysters

  • Isabelle Domart-Coulon
  • Dominique Doumenc
  • Stephanie Auzoux-Bordenave
  • Yann Le Fichant


Media supplements have been investigated for their influence on the viability of primary cell cultures from the heart ofCrassostrea gigas oysters. Soluble factors of vertebrate origin were tested, belonging to five families of supplements that had proven to increase the viability of insect and mammal cell cultures. Using two-level complete factorial assays, factors and mutual interactions were screened within each family with a MTT reduction assay. Results pointed out the positive influence of hormones, growth factor, antioxidants and lipids on the mitochondrial metabolism of oyter's heart cells. Consequently, a new concentrated complex supplement was developed. At 10% (v/v) final concentration in modified Leibovitz L-15 medium, it increases by 30% the cellular viability of one-week old cultures as compared with non-supplemented medium, a similar improvement as the one obtained with 10% (v/v) fetal calf serum. Combined with fetal calf serum, this new supplement doubles the cellular viability of one-week old cultures and allows networks of cardiomuscular cells to be maintained functional over three monthsin vitro.

Key words

Cell culture media development MTT Bivalvia oyster 



(4,5-dimethylthiazol;-2-yl)2,5-diphenyl-tetrazolium bromide


fetal calf serum


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Auzoux S, Domart-Coulon I and Doumenc D (1993) Gill cell culture of the butterfish clamRuditapes decussatus. J. Mar. Biotechnol. 1:79–81.Google Scholar
  2. Box GEP and Wilson KB (1951) On the experimental attainment of optimum conditions. J. Royal Stat. Soc. B. 13:1–45Google Scholar
  3. Brewster F and Nicholson BL (1979)In vitro maintenance of amoebocytes from the american oyster (Crassostrea virginica. J. Fish. Res. Board Can. 36:461–467.Google Scholar
  4. Brillouet C, Leclerc M, Panigel J and Binaghi R (1981)In vitro effects of various mitogens on starfish (Asterias rubens) axial organ cells. Cellular Immunology 57:136–144.Google Scholar
  5. Cecil JT (1969) Mitosis in cell cultures from cardiac tissue of the surf clamSpisula solidissima. J. Inv. Path. 14:407–410.Google Scholar
  6. Cornet M (1992) Description d'une culture de tissus de moule (Mytilus edulis destinée à la préparation de chromosomes. C. R. Acad. Sci. Sér. III 315:7–12. Cousserans F (1975) Recherches sur la culture de cellules de Mollusques marins et sur l'emploi de ces systémes cellulaires en pathologie marine. Thèse de Doctorat de 3ème cycle, Université des Sciences et Techniques du Languedoc, France.Google Scholar
  7. Coulon I (1993) Mise au point d'un système contrôlé de culture de cellules de coeur d'huîtreCrassostrea gigas. Application au test de la toxicité des produits chimiques en milieu aquatique. Thèse de Doctorat ès Sciences, Institut National Agronomique Paris-Grignon, France.Google Scholar
  8. Czech MP (1988) Signal transmission by the insulin-like growth factors. Cell 59:235–238.Google Scholar
  9. Dosuk, L (1988) Method for culturing pearls. International Patent no WO89/02919, US, priority date 28.09.87.Google Scholar
  10. Ellis LL and Bishop SH (1989) Isolation of cell lines with limited growth potential from marine bivalves. In: Mitsushashi J (Ed.) Invertebrate cell systems applications. Vol. 2(pp 243–251) CRC Press.Google Scholar
  11. Garcia-Martinez P, O'Hara S, Winston GW and Livingstone DR (1991) Oxyradical generation and redox cycling mechanisms in digestive gland microsomes of the common musselMytilus edulis L. Mar. Environ. Res. 28:271–274.Google Scholar
  12. Hamet P, Hadrava V, Kruppa U and Tremblay J (1989) Facteurs de croissance et prolifération du muscle lisse vasculaire dans l'hypertenion et le diabète. m/s 5: 654–661.Google Scholar
  13. Hervio D (1992) Contribution à l'étude deBonamia ostreae (Acetospora), protozoaire parasite de l'huîtreOstrea edulis (Bivalvia) et à l'analyse des interactions hôte-parasite. Thèse de Doctorat de l'Université Blaise Pascal de Clermont-Ferrand, France.Google Scholar
  14. Itami T, Aoki Y, Hayashi KI, Yu Y and Takahasi Y (1989)In vitro maintenance of cells of the lymphoid organ in Kuruma shrimpPenaeus japonicus. Nippon Suisan Gakkaishi 55:2205.Google Scholar
  15. Jacob F (1990) L'unité du vivant. m/s 6: 222–227.Google Scholar
  16. Kingsley RJ, Bernhardt AM, Wilbur KM and Watabe N (1987) Scleroblast cultures from the GorgonianLeptogorgia virgulata L. (Coelenterata Gorgonacea). In Vitro 23:297–302.Google Scholar
  17. Klautau M., Custodio MR and Borojevic R (1993) Cell cultures of spongesClathrina andPolymastia. In Vitro 29A:97–99.Google Scholar
  18. Kleinschuster SJ and Swink SL (1992)In vitro culture of presumptive nervous tissue ofCrassostrea virginica (Gmelin, 1791). J. Shellfish Res. 11:349–361.Google Scholar
  19. Lenoir F (1989) Mise au point de techniques de dissociation, de purification et de cultures cellulaires chez la mouleMytilus edulis L. Application à l'étude du métabolisme du glucose et du glycogène dans les cellules à glycogène (cellules vésiculeuses). Thèse de Doctorat de l'Université de Caen, France.Google Scholar
  20. Li MF, Stewart JE and Drinnan RL (1966)In vitro cultivation of cells of the oysterCrassostrea virginica. J. Fish. Res. Board Can. 23: 595–599. Machii A and Wada KT (1989) Some marine invertebrates tissue culture. In: Mitsushashi J (Ed.) Invertebrate cell systems applications. Vol. 2 (pp. 225–233) CRC Press.Google Scholar
  21. Maurer HR (1986) Towards chemically-defined, serum-free media for mammalian cell culture. In: Freshney RI (Ed.) Animal cell culture a practical approach. (pp. 13–31) IRL PressGoogle Scholar
  22. Mathieu M (1987) Etude expérimentale des contrôles exercés par les ganglions nerveux sur la gamétogénèse et les processus métaboliques associés chez la mouleMytilus edulis L. (Mollusque Lamellibranche). Thèse de Doctorat d'Etat, Université de Caen, France.Google Scholar
  23. Mialhe E, Boulo V and Grizel H (1988) Bivalve mollusc cell culture. American Fisheries Society Special Publication 18:311–315. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55–63.Google Scholar
  24. Nadal ECB, Lu Y and Loh PC (1992) Primary culture of lymphoid and nerve cells fromPenaeus stylirostris andPenaeus vannamei. In Vitro 28 (part II):86A.Google Scholar
  25. Narbonne JF (1991) Mécanismes de biotransformation des pollutants organiques chez les animaux marins. Océanis 17:449–458.Google Scholar
  26. Odintsova NA and Khomenko AV (1991) Primary cell culture from embryos of the Japanese scallopMizuchopecten yessoensis (Bivalvia). Cytotechnology 6:49–54.Google Scholar
  27. Odintsova NA and Korchagina DA (1992) Discovery of EGF-like factor in Mollusks. In Vitro 28 (part II): 86A.Google Scholar
  28. Okazaki K (1975) Spicule formation of isolated micromeres of the sea urchin embryo. Am. Zool. 15:567–581.Google Scholar
  29. Perkins FO and Menzel RW (1964) Maintenance of oyster cellsin vitro. Nature 204:1106–1107.Google Scholar
  30. Porte C, Sole M, Albaiges J and Livingstone DR (1991) Responses of mixed-function oxygenase and antioxidase enzyme system ofMytilus sp. to organic pollution. Comp. Biochem. Physiol. 100: 183–186.Google Scholar
  31. Rexen P, Kierluff JV and Emborg C (1992) An easy-to-handle semiautomated method for media development using a colorimetric viability assay and fractional factorial designs. Cytotechnology 8:195–205.Google Scholar
  32. Sami S, Ahmed II and Faisal M (1991) In vitro cultures of oysterCrassostrea virginica cells stimulation by mitogens. Chesapeake Research Consortium Publication 137:403–407.Google Scholar
  33. Swartz HM (1973) Toxic oxygen effects. Inter. Rev. Cytol. 35:321–343.Google Scholar
  34. Toullec JY, Lenoir F, Van-Wormhoudt A and Mathieu M (1988) A non-species specific growth factor from cerebral ganglia ofMytilus edulis L. J. Exp. Mar. Biol. Ecol. 119:111–117.Google Scholar
  35. Toullec JY and Porcheron P (1992) Development of primary culture of epidermal cells from the Phenaeid shrimpPenaeus vannamei. In Vitro 28 (part II):86A.Google Scholar
  36. Vago C and Chastang S (1960) Culture de tissus d'huîtres. C. R. Acad. Sci. 250:2751–2753.Google Scholar
  37. Voogt PA (1983) Lipids, their distribution and metabolism. In: Wilbur KM (ed.) The Mollusca. Vol. 1 (pp. 329–370) Academic Press, New York.Google Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Isabelle Domart-Coulon
    • 1
  • Dominique Doumenc
    • 1
  • Stephanie Auzoux-Bordenave
    • 1
  • Yann Le Fichant
    • 2
  1. 1.Laboratoire de Biologie des Invertébrés Marins CNRS URA 699Muséum National d'Histoire NaturelleParisFrance
  2. 2.Bertin & CiePlaisir CedexFrance

Personalised recommendations