Preparation of fish primary cell culture

Document Type : Full Research Paper

Author

Associate Professor of Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran.

Abstract

The viruses cause heavy losses in fish farming. Types of cell cultures, including primary cultures are appropriate tools to diagnosis and isolation the viruses. In this study with regard to the appropriate cell lines, especially for fish is not possible, or there are no suitable cells for all fish species was provided, the preparation and standardization of fish primary cell culture were done. Some samples from goldfish (Carassius auratus) internal organs including the kidney, liver, muscles, ovary and external organs including fins, snout connective tissue and gills collected and prepared according to standard procedures. The methods were used include: Enzymatic disaggregated by short and long time trypsinization and without enzyme, by planting minced tissues and ordinary method. The EMEM enriched by FBS was used for growing cells in 25oC incubation. The cells had suitable growth and reached to complete confluency and several subcultures obtained from them. The best result obtained from long time trypsinization of ovaries and muscles. Using the instructions provided in this study can be taken to prepare the primary cell culture for virus isolation and diagnosis of fish viral diseases in the country.

Keywords

Main Subjects

References


1. Akhlaghi, M. and Brojerdi, M. 1999. Anesthetic effect of clove tree and LC50 determination in rainbow trout (Oncorhynchus mykiss). Journal of Faculty of Veterinary Medicine, Univerity of Tehran 54, 49-52.
2. Chen, H., Yi, Y., Chen, M. and Yang, X. 2010. Studies on the developmental potentiality of cultured cell nuclei of fish, International Journal of Biological Sciences 6: 192–198.
3. Goff, P.L., Weil, C., Valotaire, Y., Gonnard, J.F. and Prunet, P. 1992. Effect of somatostatin on prolactin in rainbow trout (Oncorhynchus mykiss) pituitary cells in primary culture, Journal of Molecular Endocrinology 9 (2): 137-146.
4. Hong, N., Li, Z. and Hong, Y. 2011. Fish Stem Cell Cultures, International Journal of Biological Sciences 7(4): 392–402.
5. Klaunig, J.E., Rich, R. J. and Goldblatt, P.J. 1985. Trout hepatocyteculture: Isolation and primary culture, In Vitro Cellular and Developmental Biology 21 (4): 221-228.
6. Kvellestad, A., Aune, L.G. and Dannevig, B. H. 2002. Washing and disinfecting fish gills: Preventing contamination by normal surface microflora of cell cultures inoculated with tissue for isolating intracellular pathogens, Journal of Aquatic Animal Health 14 (1): 45-49.
7. Lannan, C. N., Winton, J. R. and Fryer, J. L. 1984. Fish cell lines: establishment and characterization of nine cell lines from salmonids, In Vitro 20 (9): 671-676.
8. Lee , L.E., Dayeh, V.R., Schirmer , K. and Bols, N.C. 2009. Applications and potential uses of fish gill cell lines: examples with RTgill-W1, In Vitro Cellular and Developmental Biology – Animal 45 (3-4): 127-134.
9. Marques, C.L., Rafael, M.S., Cancela, M.L. and Laize, V. 2007. Establishment of primary cell cultures from fish calcified tissues, Cytotechnology 55 (1): 9–13.
10. Nakamura, S., Kobayashi, K., Nishimura, T., Higashijima, S. and Tanaka, M. 2010. Identification of germline stem cells in the ovary of the teleost medaka, Science 328: 1561–1563.
11. Nolan, D.T., Nabben, I., Li, J. and Wendelaar Bonga S. E. 2002. Characterization of primary culture of rainbow trout (Oncorhynchus mykiss) skin explants: Growth, cell composition, proliferation and apoptosis, Society for In Vitro Biology Journal 38: 14-24.
12. Ostrander, G.K., Blair, J.B., Stark, B.A., Marley, G.M., Bales, W.D., Veltri, R., Hinton, D.E., Okihiro, M., Ortego, L.S. and Hawkins, W.E. 1995. Long-term primary culture of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver, In Vitro Cellular and Developmental Biology – Animal 31(5): 367- 378.
13. Salvo, L. M., Malucelli, M. I. C., Richartz, R. R. T. and Bacila, M. 2000. Primary culture of hepatic cells from Metynnis roosevelti (Pisces, Teleostei, Characidae), Brazilian Journal of Veterinary Research and Animal Science 37(5): 1-11.
14. Scholz, S., Braunbeck, T. and Segner, H. 1998. Viability and differential function of rainbow trout liver cells in primary culture: Coculture with two permanent fish cells, In Vitro Cellular and Developmental Biology – Animal 34: 762-771.
15. Stott, L.C., Schnell, S., Hogstrand, C., Owen, S.F. and Bury, N.R. 2015. A primary fish gill cell culture model to assess pharmaceutical uptake and efflux: Evidence for passive and facilitated transport, Aquatic Toxicology 159: 127–137.
16. Wolf, K. and Quimby, M. C. 1976. Primary Monolayer Culture of Fish Cells Initiated from Tripsinized Tissues, TCA Manual Vol 2, No 4.
17. Wood, C.M., Eletti, B. and Part, P. 2002. New methods for the primary culture of gill epithelia from freshwater rainbow trout, Fish Physiology and Biochemistry 26 (4): 329-344.
18. Yi, M., Hong, N. and Hong, Y. 2010. Derivation and characterization of haploid embryonic stem cell cultures in medaka fish, Nature Protocols 5:1418–1430.

Files

History

  • Receive Date: 23 August 2017
  • Revise Date: 02 October 2017
  • Accept Date: 02 October 2017

Share

How to cite

Statistics