, Volume 67, Issue 3, pp 559–571 | Cite as

Multifactorial interaction of growth factors on Penaeus monodon lymphoid cells and the impact of IGFs in DNA synthesis and metabolic activity in vitro

  • P. Jayesh
  • Rosamma Philip
  • I. S. Bright Singh
Method in Cell Science


Development of continuous cell lines from shrimp is essential to investigate viral pathogens. Unfortunately, there is no valid cell line developed from crustaceans in general and shrimps in particular to address this issue. Lack of information on the requirements of cells in vitro limits the success of developing a cell line, where the microenvironment of a cell culture, provided by the growth medium, is of prime importance. Screening and optimization of growth medium components based on statistical experimental designs have been widely used for improving the efficacy of cell culture media. Accordingly, we applied Plackett–Burman design and response surface methodology to study multifactorial interactions between the growth factors in shrimp cell culture medium and to identify the most important ones for growth of lymphoid cell culture from Penaeus monodon. The statistical screening and optimization indicated that insulin like growth factor-I (IGF-I) and insulin like growth factor-II (IGF-II) at concentrations of 100 and 150 ng ml−1, respectively, could significantly influence the metabolic activity and DNA synthesis of the lymphoid cells. An increase of 53 % metabolic activity and 24.8 % DNA synthesis could be obtained, which suggested that IGF-I and IGF-II had critical roles in metabolic activity and DNA synthesis of shrimp lymphoid cells.


Growth factor optimization Response surface methodology Plackett–Burman Central composite design Penaeus monodon Lymphoid cell culture Shrimp cell culture medium 



This research was supported by Department of Biotechnology, Government of India (BT/PR8050/AAQ/03/289/2006). The first author thanks DBT for Fellowship.

Supplementary material

10616_2014_9697_MOESM1_ESM.doc (352 kb)
Supplementary material 1 (DOC 352 kb)


  1. Ahuja SK, Ferreira GM, Moreira AR (2004) Application of Plackett-Burman design and response surface methodology to achieve exponential growth for aggregated shipworm bacterium. Biotechnol Bioeng 85:666–675CrossRefGoogle Scholar
  2. Baker PF, Carruthers A (1980) Insulin stimulates sugar transport in giant muscle fibres of the barnacle. Nature 286:276–279CrossRefGoogle Scholar
  3. Bell TA, Lightner DV (1988) A handbook of normal penaeid shrimp histology. Special Publication No. 1. World Aquac Soc 52–73Google Scholar
  4. Bonami JR, Lightner DV, Redman RM, Poulos BT (1992) Partial characterisation of a togavirus (LOVV) associated with histopathological changes of the lymphoid organ of penaeid shrimps. Dis Aquat Org 14:145–152CrossRefGoogle Scholar
  5. Box GEP, Wilson KB (1951) On the experimental attainment of optimum conditions. J Roy Stat Soc 13:1–45Google Scholar
  6. Braasch DA, Ellender RD, Middlebrooks BL (1999) Cell cycle components and their potential impact on the development of continuous in vitro penaeid cell replication. Methods Cell Sci 21:255–261CrossRefGoogle Scholar
  7. Castro PML, Hayter PM, Ison AP, Bull AT (1992) Application of a statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells. Appl Microbiol Biotechnol 38:84–90Google Scholar
  8. Cervera L, Gutiérrez-Granados S, Martínez M, Blanco J, Gòdia F, Segura MM (2013) Generation of HIV-1 Gag VLPs by transient transfection of HEK 293 suspension cell cultures using an optimized animal-derived component free medium. J Biotechnol 166:152–165CrossRefGoogle Scholar
  9. Chantanachookin C, Boonyaratpalin S, Kasornchandra J, Direkbusarakom S, Ekpanithanpong U, Supamataya K, Sriurairatana S, Flegel TW (1993) Histology and ultrastructure reveal a new granulosis-like virus in Penaeus monodon affected by yellow-head disease. Dis Aquat Org 17:145–157CrossRefGoogle Scholar
  10. Chaulet A, Medesani DA, Freitas J, Cervino A, Cervino N, Rodríguez EM (2012) Induction of somatic growth in juvenile crayfish Cherax quadricarinatus (Decapoda, Parastacidae), by ecdysone and insulin growth factor. Aquaculture 370–371:1–6CrossRefGoogle Scholar
  11. Chen SN, Kou GH (1989) Infection of cultured cells from lymphoid organ of Penaeus monodon Fabricius by monodon-type baculovirus (MBV). J Fish Dis 12:73–76CrossRefGoogle Scholar
  12. Fan TJ, Wang XF (2002) In vitro culture of embryonic cells from the shrimp, P. chinensis. J Exp Mar Biol Eco 267:175–184CrossRefGoogle Scholar
  13. Fraser CA, Owens L (1996) Spawner-isolated mortality virus from Australian Penaeus monodon. Dis Aquat Org 27:141–148CrossRefGoogle Scholar
  14. Freshney RI (2000) Culture of animal cells: a manual of basic technique. Wiley-Liss, New YorkGoogle Scholar
  15. George SK, Dhar AK (2010) An improved method of cell culture system from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of Penaeus vannamei. In Vitro Cell Dev - An 46:801–810CrossRefGoogle Scholar
  16. González-Leal IJ, Carrillo-Cocom LM, Ramírez-Medrano A, López-Pacheco F, Bulnes-Abundis D, Webb-Vargas Y, Alvarez MM (2011) Use of a Plackett–Burman statistical design to determine the effect of selected amino acids on monoclonal antibody production in CHO cells. Biotechnol Prog 27:1709–1717CrossRefGoogle Scholar
  17. Gratzner HJ (1982) Monoclonal antibody to 5-bromo and 5-iododeoxyuridine: a new reagent for the detection of DNA replication. Science 218:474–475CrossRefGoogle Scholar
  18. Guilford JP, Fruchter B (1973) Fundamental statistics in psychology and education. McGraw-Hill, New YorkGoogle Scholar
  19. Gutiérrez A, Nieto J, Pozo F, Stern S, Schoofs L (2007) Effect of insulin/IGF-I like peptides on glucose metabolism in the white shrimp Penaeus vannamei. Gen Comp Endocrinol 153:170–175CrossRefGoogle Scholar
  20. Haaland PD (1989) Separating signals from the noise. Experimental design in biotechnology. Marcel Dekker, New York, pp 61–83Google Scholar
  21. Hasson KW, Lightner DV, Mohney LL, Redman RM, White BM (1999) Role of lymphoid organ spheroids in chronic Taura syndrome virus (TSV) infections in Penaeus vannamei. Dis Aquat Org 38:93–105CrossRefGoogle Scholar
  22. 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
  23. Jayesh P (2013) Development of lymphoid cell culture system from Penaeus monodon and molecular approaches for its transformation. Ph.D. Thesis. Cochin University of Science and Technology, IndiaGoogle Scholar
  24. Jayesh P, Seena J, Singh ISB (2012) Establishment of shrimp cell lines: perception and orientation. Indian J Virol 23:244–251CrossRefGoogle Scholar
  25. Jayesh P, Seena J, Philip R, Singh ISB (2013) A novel medium for the development of in vitro cell culture system from Penaeus monodon. Cytotechnology 65:307–322CrossRefGoogle Scholar
  26. Jose S, Mohandas A, Philip R, Singh ISB (2010) Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays. J Invertebr Pathol 105:312–321CrossRefGoogle Scholar
  27. Jose S, Jayesh P, Mohandas A, Philip R, Singh ISB (2011) Application of primary haemocyte culture of Penaeus monodon in the assessment of cytotoxicity and genotoxicity of heavy metals and pesticides. Mar Environ Res 71:169–177CrossRefGoogle Scholar
  28. Jose S, Jayesh P, Sudheer NS, Poulose G, Mohandas A, Philip R, Bright ISB (2012) Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression. J Fish Dis 35:321–334CrossRefGoogle Scholar
  29. Kumar VJR, Achuthan C, Manju NJ, Philip R, Singh ISB (2009) Activated packed bed bioreactor for rapid nitrification in brackish water hatchery systems. J Ind Microbiol Biot 36:355–365CrossRefGoogle Scholar
  30. Kumar VJR, Joseph V, Vijai R, Philip R, Singh ISB (2011) Nitrification in a packed bed bioreactor integrated into a marine recirculating maturation system under different substrate concentrations and flow rates. J Chem Technol Biotechnol 86:790–797CrossRefGoogle Scholar
  31. Lee GM, Kim EJ, Kim NS, Yoon SK, Ahn YH, Song JY (1999) Development of a serum-free medium for the production of erythropoietin by suspension culture of recombinant Chinese hamster ovary cells using a statistical design. J Biotechnol 69:85–93CrossRefGoogle Scholar
  32. Mathews LS, Hammer RE, Behringer RR, D’Ercole AJ, Bell GI, Brinster RL, Palmiter RD (1988) Growth enhancement of transgenic mice expressing human insulin-like growth factor-I. Endocrinology 123:2827–2833CrossRefGoogle Scholar
  33. Maeda M, Mizuki E, Itami T, Ohba M (2003) Ovarian primary tissue culture of the kuruma shrimp Marsupenaeus japonicus. In Vitro Cell Dev-An 39:208–212Google Scholar
  34. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival, application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63CrossRefGoogle Scholar
  35. Mulford AL, Lyng F, Mothersill C, Austin B (2001) Development and characterization of primary cell cultures from the hematopoietic tissues of the Dublin Bay prawn, Nephrops norvegicus. Methods Cell Sci 22:265–275CrossRefGoogle Scholar
  36. Nadala EC, Loh PC, Lu PC (1993) Primary culture of lymphoid, nerve and ovary cells from Penaeus stylirostris and Penaeus vannamei. In Vitro Cell Dev-An 29:620–622CrossRefGoogle Scholar
  37. Nadala ECB, Lu Y, Loh PC, Brock JA (1992) Infection of Penaeus stylirostris (Boone) with a rhabdovirus isolated from Penaeus spp. Gyobyo Kenkyu 27:143–147Google Scholar
  38. Owens L, Beer SD, Smith J (1991) Lymphoidal parvovirus-like particles in Australian penaeid prawns. Dis Aquat Org 11:129–134CrossRefGoogle Scholar
  39. Plackett RL, Burman JP (1946) The design of optimum multifactorial experiments. Biometrika 33:305–325CrossRefGoogle Scholar
  40. Rajendran KV, Cowley JA, McCulloch RJ, Walker PJ (2006) A TaqMan real-time RTPCR for quantifying Mourilyan virus infection levels in penaeid shrimp tissues. J Virol Methods 137:265–271CrossRefGoogle Scholar
  41. Richardson NA, Anderson AJ, Sara VR (1997) The effects of insulin/IGF-I on glucose and leucine metabolism in the redclaw crayfish (Cherax quadricarinatus). Gen Comp Endocrinol 105:287–293CrossRefGoogle Scholar
  42. Rodríguez J, Bayot B, Amano Y, Panchana F, Blas I, Alday V, Calderón J (2003) White spot syndrome virus infection in cultured Penaeus vannamei (Boone) in Ecuador with emphasis on histopathology and ultrastructure. J Fish Dis 26:439–450CrossRefGoogle Scholar
  43. Rusaini, Owens L (2010) Insight into the lymphoid organ of penaeid prawns: a review. Fish Shellfish Immunol 29:367–377Google Scholar
  44. Spann KM, Vickers JE, Lester RJG (1995) Lymphoid organ virus of Penaeus monodon from Australia. Dis Aquat Org 23:127–134CrossRefGoogle Scholar
  45. Sritunyalucksana K, Apisawetakan S, Boon-nat A, Withyachumnarnkul B, Flegel TW (2006) A new RNA virus found in black tiger shrimp Penaeus monodon from Thailand. Virus Res 118:31–38CrossRefGoogle Scholar
  46. Stanbury PF, Whitaker A, Hall SJ (1986) Media for industrial fermentations. Principles of fermentation technology. Pergamon, Oxford, pp 93–122Google Scholar
  47. Tang KFJ, Pantoja CJ, Poulos BT, Redman RM, Lightner DV (2005) In situ hybridization demonstrates that Litopenaeus vannamei, L. stylirostris and Penaeus monodon are susceptible to experimental infection with infectious myonecrosis virus (IMNV). Dis Aquat Org 63:261–265CrossRefGoogle Scholar
  48. Tapay L, Lu Y, Brock JA, Nadala ECB, Loh PC (1995) Transformation of primary cultures of shrimp (Penaeus stylirostris) lymphoid (Oka) organ with simian virus-40 (T) antigen. Proc Soc Exp Biol Med 209:73–78CrossRefGoogle Scholar
  49. Wang Y-X, Lu Z-X (2004) Statistical optimization of media for extracellular polysaccharide by Pholiota squarrosa (Pers. ex Fr.) Quel. AS 5.245 under submerged cultivation. Biochem Eng J 20:39–47CrossRefGoogle Scholar
  50. Wang C-H, Yang H-N, Tang C-Y, Lu CH, Kou GH, Lo C-F (2000) Ultrastructure of white spot syndrome virus development in primary lymphoid organ cell cultures. Dis Aquat Organ 41:91–104CrossRefGoogle Scholar
  51. Xia S, Laterra J (2006) Hepatocyte growth factor increases mitochondrial mass in glioblastoma cells. Biochem Bioph Res Commun 345:1358–1364CrossRefGoogle Scholar
  52. Zhang H, Wang H, Liu M, Zhang T, Zhang J, Wang X, Xiang W (2013) Rational development of a serum-free medium and fed-batch process for a GS-CHO cell line expressing recombinant antibody. Cytotechnology 65:363–378CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • P. Jayesh
    • 1
  • Rosamma Philip
    • 2
  • I. S. Bright Singh
    • 1
  1. 1.National Centre for Aquatic Animal HealthCochin University of Science and TechnologyKochiIndia
  2. 2.Department of Marine Biology, Microbiology and BiochemistryCochin University of Science and TechnologyKochiIndia

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