Advertisement

Fish Physiology and Biochemistry

, Volume 36, Issue 4, pp 1181–1189 | Cite as

Establishment, characterization of a new cell line from heart of half smooth tongue sole (Cynoglossus semilaevis)

  • X. L. Wang
  • N. Wang
  • Z. X. Sha
  • S. L. Chen
Article

Abstract

A new cell line was established from the heart of a cultured marine fish, half smooth tongue sole (Cynoglossus semilaevis), designated as CSH (Cynoglossus semilaevis heart cell line). The CSH cells grow over 400 days in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 2 ng/ml basic fibroblast growth factor (bFGF). The suitable temperature for the cell growth was 24–30°C with the optimum growth at 24°C and a reduced growth at 12 and 30°C. FBS and bFGF concentration were the two important components for CSH cells proliferation. Twenty percent FBS in the medium was found to be the optimum concentration and bFGF promoted the growth of CSH cells. The double time of the cells at 24°C was determined to 73.39 h. Chromosome analysis revealed that 44% of the cells maintained a normal diploid chromosome number (2n = 42) in the CSH cells at Passage 58. The fluorescent signals were observed in CSH after the cells were transfected with green fluorescent protein (GFP) reporter plasmids. CSH cells showed the cytopathic effect (CPE) after infection with lymphosystis disease virus (LCDV). Moreover, the LCDV particles can be observed in the cytoplasm of virus-infected cells by electron microscopy, and a segment of MCP gene for major capsid protein of LCDV was found by PCR amplification DNA of virus-infected cells.

Keywords

Cell line Half smooth tongue sole (Cynoglossus semilaevisCSH Karyotype GFP LCDV 

Notes

Acknowledgments

This work was supported by grants from State 863 High-Technology R&D Project of China (2006AA09Z406, 2006AA10A401), and Taishan Scholar Project of Shandong Province.

References

  1. Alonso MC, Ferro P, Garcia-Rosado E, Cano I, Lang T, Bergmann SM, Borrego JJ (2007) Comparison of lymphocystis disease virus (LCDV) isolates obtained from different marine fish species and geographical areas. Bull Eur Assoc Fish Pathol 27:157–164Google Scholar
  2. Bahich H, Borenfreund E (1991) Cytotoxicity and genotoxicity assays with cultured fish cells: a review. Toxic Vitro 5:91–100CrossRefGoogle Scholar
  3. Bejar J, Porta J, Borrego JJ, Alvarez MC (2005) The piscine SAF-1 cell line: genetic stability and labeling. Mar Biotech 7:389–395CrossRefGoogle Scholar
  4. Bols NC, Dayeh VR, Lee LEJ, Schirmer K (2005) Use of fish cell lines in the toxicology and ecotoxicology of fish. In: Moon TW, Mommsen TP (eds) Biochemistry and molecular biology of fishes -environmental toxicology, vol. 6. Elsevier, Amsterdam, pp 43–84Google Scholar
  5. Bradford CS, Sun L, Barnes DW (1994) Basic fibroblast growth factor stimulates proliferation and suppresses melanogenesis in cell cultures derived from early zebrafish embryos. Mol Mar Biol Biotech 3:78–86Google Scholar
  6. Chen SC, Hu WW, Lo BJ (1999) Establishment and characterization of a continuous cell line (GF-1) derived from grouper, Epinephelus coioides: a cell line susceptible to grouper nervous necrosis virus (GNNV). J Fish Dis 22:173–182CrossRefGoogle Scholar
  7. Chen SL, Ye HQ, Sha ZX, Hong Y (2003a) Derivation of a pluripotent embryonic cell line from red sea bream blastulas. J Fish Biol 63:795–805CrossRefGoogle Scholar
  8. Chen SL, Sha ZX, Ye HQ (2003b) Establishment of a pluripotent embryonic cell line from sea perch (Lateolabrax japonicus) blastula embryo. Aquaculture 218:141–151CrossRefGoogle Scholar
  9. Chen SL, Ren GC, Sha ZX, Shi CY (2004) Establishment of a continuous embryonic cell line from Japanese flounder for virus isolation. Dis Aqua Org 60:241–246CrossRefGoogle Scholar
  10. Chen SL, Sha ZX, Ye HQ, Liu Y, Tian YS, Hong Y, Tang QS (2007) Pluripotency and chimera competence of an embryonic stem cell line from sea perch (Lateolabrax japonicus). Mar Biotech 9:82–91CrossRefGoogle Scholar
  11. Freshney RI (1994) Culture of animal cells. In: Freshney RI (ed) A manual of basic techniques. Wiley, New York, pp 387–389Google Scholar
  12. Fryer JL, Lannan CN (1994) Three decades of fish cell culture: a current listing of cell lines derived from fishes. J Tiss Cult Meth 16:87–94CrossRefGoogle Scholar
  13. Halaban R, Langdon R, Birchall N, Cuono C, Baird A, Scott G, Moellman G, McGuire J (1988) Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes. J Cell Biol 107:1611–1619CrossRefPubMedGoogle Scholar
  14. Hayflick L (1973) Theory of population increase by subcultivation. In: Kruse PF, Patterson MK (eds) Tissue culture methods and application. Academic Press, NY, pp 222–223Google Scholar
  15. Hightower LH, Renfro JL (1988) Recent applications of fish cell culture to biomedical research. J Exp Zool 248:290–302CrossRefPubMedGoogle Scholar
  16. Hong Y, Schartl M (1996) Establishment and growth responses of early medaka fish embryonic cells in feeder layer-free cultures. Mol mar biol biotech 5:93–104Google Scholar
  17. Hsu YL, Yang YH, Chen YC, Tung MC, Wu JL, Engelking MH, Leong JC (1995) Development of an in vitro subculture system for the oka organ (Lymphoid tissue) of Penaeus monodon. Aquaculture 136:43–55CrossRefGoogle Scholar
  18. Lee LEJ, Dayeh VR, Schirmer K, Bols NC (2009) Application and potential uses of fish gill cell lines: examples with RTgill-W1. In Vitro Cell Dev Biol Anim 45:127–134CrossRefPubMedGoogle Scholar
  19. Levan A (1964) Nomenclature for centromeric position on chromosomes. Hereditas 52:210–220Google Scholar
  20. Matsui Y, Zsebo K, Hogan BLM (1992) Derivation of pluripotential embryonic stem cell line from murine primordial germ cells in culture. Cell 70:41–847CrossRefGoogle Scholar
  21. Nicholson BL, Danner DJ, Wu JL (1987) Three new continuous fish continuous cell lines from marine fishes of Asia. Vitro Cell Dev Biol 23:199–204CrossRefGoogle Scholar
  22. Parameswaran V, Ishaq Ahmed VP, Ravi S, Bhonde RR, Sahul Hameed AS (2007a) Development and characterization of two new cell lines from milkfish (Chanos chanos) and grouper (Epinephelus coioides) for virus isolation. Mar Biotechnol 9:281–291CrossRefPubMedGoogle Scholar
  23. Parameswaran V, Shukla R, Blonde R, Hameed AS (2007b) Development of a pluripotent ES-like cell line from Asian sea bass (Lates calcarifer)—an oviparous stem cell line mimicking viviparous ES cells. Mar Biotechnol 9:766–775CrossRefPubMedGoogle Scholar
  24. Ren GC, Chen SL, Sha ZX (2008) Development and characterization of a liver cell line from half-smooth tongue sole (Cynoglossus semilaevis). Chin High Technol Lett 18(6):657–660Google Scholar
  25. Segner H (1998) Fish cell lines as a tool in aquatic toxicology. In: Braunbeck T, Hinton DE, Streit B (eds) Fish ecotoxicology. Birkhäuser, Basel, pp 1–38Google Scholar
  26. Sha ZX, Ren GC, Wang XL, Wang N, Chen SL (2010) Development and characterization of a cell line from the embryos of half smooth tongue sole (Cynoglossus semilaevis). Chin Acta Oceanologica Sinica (in press)Google Scholar
  27. Villena AJ (2003) Applications and needs of fish and shellfish cell culture for disease control in aquaculture. Rev Fish Biol Fisher 13:111–140CrossRefGoogle Scholar
  28. Wan RJ, Jiang YW, Zhuang ZM (2004) Morphological and development characters at the early stages of the tonguefish Cymoglossus semilaevis. Acta Zool Sinlca 50(1):91–102Google Scholar
  29. Weissenberg R (1965) 50 Years of research on the lymphocystis virus disease of fishes (1914–1964). Ann N Y Acad Sci 126:362–374CrossRefPubMedGoogle Scholar
  30. Xu HT, Piao CA, Jiang ZL, Wang WX (2000) Study on the causative agent of lymphocystic disease in cultured flounder, Paralichthys Olivaceus, in mainland. China Chin Virol 16:223–226Google Scholar
  31. Ye HQ, Chen SL, Sha ZX, Xu MY (2006) Development and characterization of cell lines from heart, liver, spleen and head kidney of sea perch Lateolabrax japonicus. J Fish Biol (Supplement A) 69:115–126CrossRefGoogle Scholar
  32. Zhou Lq, Yang Ag, Liu XZ, Du W, ZM Zhuang (2005) The karyotype of the tonguefish Cynoglossus semilaevis. J Fish China 29:417–419Google Scholar
  33. Zhou GZ, Li ZQ, Yuan XP, Zhang QY (2007) Establishment, characterization, and virus susceptibility of a new marine cell line from red spotted grouper (Epinephelus akaara). Mar Biotech 9:370–376CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • X. L. Wang
    • 1
    • 2
  • N. Wang
    • 1
  • Z. X. Sha
    • 1
  • S. L. Chen
    • 1
  1. 1.Key Lab for Sustainable Utilization of Marine Fisheries Resources, Ministry of Agriculture, Yellow Sea Fisheries Research InstituteChinese Academy of Fishery SciencesQingdaoChina
  2. 2.College of Marine Life ScienceOcean University of ChinaQingdaoChina

Personalised recommendations