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. 2003 Sep;42(3):109–120. doi: 10.1023/B:CYTO.0000015795.46813.44

Growth, viral production and metabolism of a Helicoverpa zea cell line in serum-free culture

Linda HL Lua 1,✉, Steven Reid 1
PMCID: PMC3449452  PMID: 19002933

Abstract

Insect cell cultures have been extensively utilised for means of production for heterologous proteins and biopesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five™) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines have been investigated thoroughly over recent years. The Helicoverpa zea cell line has potential use for the production of a biopesticide, specifically the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). The growth, virus production, nutrient consumption and waste production of this cell line was investigated under serum-free culture conditions, using SF900II and a low cost medium prototype (LCM). The cell growth (growth rates and population doubling time) was comparable in SF900II and LCM, however, lower biomass and cell specific virus yields were obtained in LCM. H. zea cells showed a preference for asparagine over glutamine, similar to the High Five™ cells. Ammonia was accumulated to significantly high levels (16 mM) in SF900II, which is an asparagine and glutamine rich medium. However, given the absence of asparagine and glutamine in the medium (LCM), H. zea cells adapted and grew well in the absence of these substrates and no accumulation of ammonia was observed. The adverse effect of ammonia on H. zea cells is unknown since good production of biologically active HaSNPV was achieved in the presence of high ammonia levels. H. zea cells showed a preference for maltose even given an abundance supply of free glucose. Accumulation of lactate was observed in H. zea cell cultures.

Keywords: Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus, Helicoverpa zea, Serum-free low cost medium

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References

  1. Agathos S.N. Mass production of viral insecticides. In: Maramorosch K, editor. Biotechnology for Biological Control of Pests and Vectors. Florida: CRC Press; 1991. pp. 217–235. [Google Scholar]
  2. Bedard C., Kamen A., Tom R., Massie B. Maximisation of recombinant protein yield in the insect cell/ baculovirus system by one time addition of nutrients to highdensity batch cultures. Cytotechnology. 1994;15:129–138. doi: 10.1007/BF00762387. [DOI] [PubMed] [Google Scholar]
  3. Bedard C., Kamen A.A. Metabolism of insect cells cultured in vitro. In: Maramorosch K., Mitsuhashi J, editors. Invertebrate Cell Culture. USA: Novel Directions and Biotechnology Applications, Science Publishers; 1997. pp. 35–42. [Google Scholar]
  4. Bedard C., Perret S., Kamen A.A. Fed-batch culture of Sf-9 cells supports 3 × 107 cells per ml and improves baculovirus-expressed recombinant protein yields. Biotechnol. Lett. 1997;19:629–632. doi: 10.1023/A:1018378529299. [DOI] [Google Scholar]
  5. Bedard C., Tom R., Kamen A., Andre G. Nutrient consumption and waste product accumulation in Sf9 insect cell line. In: Vlak J. S. E. A., editor. Baculovirus and Recombinant Protein Production Processes. Switzerland: Editions Roche; 1992. pp. 339–360. [Google Scholar]
  6. Bedard C., Tome R., Kamen A. Growth, nutrient consumption, and the end-product accumulation in Sf9 and BTI-EEA insect cell cultures: insights into growth limitation and metabolism. Biotechnol. Prog. 1993;9:615–624. doi: 10.1021/bp00024a008. [DOI] [PubMed] [Google Scholar]
  7. Chakraborty S., Greenfield P.F., Reid S. In vitro production studies with a wild-type Helicoverpa baculovirus. Cytotechnology. 1996;22:217–224. doi: 10.1007/BF00353942. [DOI] [PubMed] [Google Scholar]
  8. Chakraborty S., Kanhaisingh A., Greenfield P.F., Reid S., Monsour C.J., Teakle R. In vitro production of wild type Heliothis baculoviruses for use as biopesticides. Aust. Biotechnol. 1995;5:82–86. [Google Scholar]
  9. Chakraborty S., Monsour C., Teakle R., Reid S. Yield, biological activity, and field performance of a wild-type Helicoverpa nucleopolyhedrovirus produced in Helicoverpa zea cell cultures. J. Invertebr. Pathol. 1999;73:199–205. doi: 10.1006/jipa.1998.4825. [DOI] [PubMed] [Google Scholar]
  10. Chakraborty S., Reid S. Serial passage of a Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea cell cultures. J. Invertebr. Pathol. 1999;73:303–308. doi: 10.1006/jipa.1999.4848. [DOI] [PubMed] [Google Scholar]
  11. Chen X., Ijkel F.W.J., Tarchini R., Sun X., Sandbrink H., Wang H., Peters S., Zuidema D., Lankhorst R.K., Vlak J.M., Hu Z. The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome. J. Gen. Virol. 2001;82:241–257. doi: 10.1099/0022-1317-82-1-241. [DOI] [PubMed] [Google Scholar]
  12. Chen X., Sun X., Hu Z., Li M., O'Reilly D.R., Zuidema D., Vlak J.M. Genetic engineering of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus as an improved pesticide. J. Invertebr. Pathol. 2000;76:140–146. doi: 10.1006/jipa.2000.4963. [DOI] [PubMed] [Google Scholar]
  13. Chen X., Zhang W.-J., Wong J., Chun G., Lu A., McCutchen B.F., Presnail J.K., Herrmann R., Dolan M., Tingey S., Hu Z.H., Vlak J.M. Comparative analysis of the complete genome sequences of Helicoverpa zea and Helicoverpa armigera single-nucleocapsid nucleopolyhedroviruses. J. Gen. Virol. 2002;83:673–684. doi: 10.1099/0022-1317-83-3-673. [DOI] [PubMed] [Google Scholar]
  14. Donaldson M.S., Shuler M.L. Effects of long-term passaging of BTI-Tn5B1-4 insect cells on growth and recombinant protein production. Biotechnol. Prog. 1998;14:543–547. doi: 10.1021/bp9800485. [DOI] [PubMed] [Google Scholar]
  15. Donaldson M.S., Shuler M.L. Low cost serum-free medium for the BTI-Tn5B1-4 insect cell line. Biotechnol. Prog. 1998;14:573–579. doi: 10.1021/bp9800541. [DOI] [PubMed] [Google Scholar]
  16. Doyle C., Butler M. The effect of pH on the toxicity of ammonia to a murine hybridoma. J. Biotechnol. 1990;15:91–100. doi: 10.1016/0168-1656(90)90053-E. [DOI] [PubMed] [Google Scholar]
  17. Ferrance P., Goel A., Ataai M.M. Utilisation of glucose and amino acids in insect cell cultures: quantifying the metabolic flows within the primary pathways and medium development. Biotechnol. Bioeng. 1993;42:697–707. doi: 10.1002/bit.260420604. [DOI] [PubMed] [Google Scholar]
  18. Ghosh S., Parvez M.K., Banerjee K., Sarin S.K., Hasnain S.E. Baculovirus as mammalian cell expression vector for gene therapy: an emerging strategy. Mol. Ther. 2002;6:5–11. doi: 10.1006/mthe.2000.0643. [DOI] [PubMed] [Google Scholar]
  19. Gilbert R.S., Nagano Y., Yokota T., Fletcher T., Lydersen K. Effect of lipids on insect cell growth and expression of recombinant proteins in serum-free medium. Cytotechnology. 1996;22:211–216. doi: 10.1007/BF00353941. [DOI] [PubMed] [Google Scholar]
  20. Goodman C.L., McIntosh A.H. Production of baculoviruses for insect control using cell culture. In: Maramorosch K., McIntosh A.H, editors. Insect Cell Biotechnology. Florida: CRC Press; 1994. pp. 33–56. [Google Scholar]
  21. Granados R.R., Lawler K.A., Burand J.P. Replication of Heliothis zea baculovirus in an insect cell line. Intervirology. 1981;16:71–79. doi: 10.1159/000149250. [DOI] [PubMed] [Google Scholar]
  22. Granados R.R., McKenna K.A. Insect cell culture methods and their use in virus research. In: Shuler M.L., Wood H.A., Granados R.R., Hammer D.A, editors. Baculovirus Expression Systems and Biopesticides. New York: Wiley-Liss, Inc.; 1995. pp. 13–39. [Google Scholar]
  23. Hassell T., Gleave S., Butler M. Growth inhibition in animal cell culture. Appl. Biochem. Biotechnol. 1991;30:29–41. doi: 10.1007/BF02922022. [DOI] [PubMed] [Google Scholar]
  24. Ignoffo C.M., Shapiro M., Hink W.F. Replication and serial passage of infectious Heliothis nucleopolyhedrosis virus in an established line of Heliothis zea cells. J. Invertebr. Pathol. 1971;18:131–134. doi: 10.1016/0022-2011(91)90021-H. [DOI] [PubMed] [Google Scholar]
  25. Le T., Robertson A., Bulach D., Cowan P., Goodge K., Tribe D. Genetically variable triplet repeats in a RING-finger ORF of Helicoverpa species baculoviruses. Virus Res. 1997;49:67–77. doi: 10.1016/S0168-1702(97)01454-8. [DOI] [PubMed] [Google Scholar]
  26. Lenz C.J., McIntosh A.H., Mazzacano C., Monderloh U. Replication of Heliothis zea nuclear polyhedrosis virus in cloned cell lines. J. Invertebr. Pathol. 1991;57:227–233. doi: 10.1016/0022-2011(91)90121-6. [DOI] [PubMed] [Google Scholar]
  27. Lua L.H.L., Nielsen L.K., Reid S. Sensitivity of Helicoverpa armigera nucleopolyhedrovirus to sodium dodecyl sulfate (SDS) Biol. Control. 2003;26:57–67. doi: 10.1016/S1049-9644(02)00116-0. [DOI] [Google Scholar]
  28. Lua L.H.L., Pedrini M., Reid S., Robertson A., Tribe D.E. Phenotypic and genotypic analysis of Helicoverpa armigera nucleopolyhedrovirus serially passaged in cell culture. J. Gen. Virol. 2002;83:947–957. doi: 10.1099/0022-1317-83-4-945. [DOI] [PubMed] [Google Scholar]
  29. Lua L.H.L., Reid S. Virus morphogenesis of Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea serum-free suspension culture. J. Gen. Virol. 2000;81:2531–2543. doi: 10.1099/0022-1317-81-10-2531. [DOI] [PubMed] [Google Scholar]
  30. Lua L.H.L., Reid S. Effect of time of harvest of budded virus on the selection of baculovirus FP mutants in cell culture. Biotechnol. Prog. 2003;19:238–242. doi: 10.1021/bp025577u. [DOI] [PubMed] [Google Scholar]
  31. Marheineke K., Grunewald S., Christie W., Reilander H. Lipid composition of Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) insect cells used for baculovirus infection. FEBS Lett. 1998;441:49–52. doi: 10.1016/S0014-5793(98)01523-3. [DOI] [PubMed] [Google Scholar]
  32. Marteijn R.C., Jurrius O., Dhont J., De Gooijer C.D., Tramper J., Martens D.E. Optimization of a feed medium for fed-batch culture of insect cells using a genetic algorithm. Biotechnol. Bioeng. 2003;81:269–278. doi: 10.1002/bit.10465. [DOI] [PubMed] [Google Scholar]
  33. Martinelle K., Westlund A., Haggstrom L. Ammonium ion transport - a cause of cell death. Cytotechnology. 1996;22:251–254. doi: 10.1007/BF00353945. [DOI] [PubMed] [Google Scholar]
  34. McIntosh A.H., Goodman C.L., Grasela J.J. Virus production and plaque-forming ability of Helicoverpa zea (Lepidoptera: Noctuidae) cell line. Appl. Entomol. Zool. 1997;32:358–657. [Google Scholar]
  35. McIntosh A.H., Grasela J.J., Goodman C.L. Replication of Helicoverpa zea nuclear polyhedrosis virus in homologous cell lines grown in serum-free media. J. Invertebr. Pathol. 1995;66:121–124. doi: 10.1006/jipa.1995.1073. [DOI] [Google Scholar]
  36. McIntosh A.H., Grasela J.J., Goodman C.L., Ignoffo C.M. Growth of a clonal line of Helicoverpa zea (Lepidoptera: noctuidae) in suspension culture and replication of its homologous baculovirus HzSNPV. Appl. Entomol. Zool. 2001;36:349–352. doi: 10.1303/aez.2001.349. [DOI] [Google Scholar]
  37. McIntosh A.H., Ignoffo C.M. Replication and infectivity of the single-embedded nuclear polyhedrosis virus, baculovirus Heliothis, in homologous cell line. J. Invertebr. Pathol. 1981;37:258–264. doi: 10.1016/0022-2011(81)90084-7. [DOI] [Google Scholar]
  38. Metzler D.E. Biochemistry: The Chemical Reactions of Living Cells. New York: Academic Press; 1977. [Google Scholar]
  39. Miller L.K., Lu A. The molecular basis of baculovirus host range. In: Miller L.K, editor. The Baculoviruses. New York: Plenum Press; 1997. pp. 217–235. [Google Scholar]
  40. Mitsuhashi J. Media for insect cell cultures. Adv. Cell Cult. 1982;2:133–196. [Google Scholar]
  41. Mitsuhashi J. Nutritional requirements of insect cells in vitro. In: Mitsuhashi J, editor. Invertebrate Cell System Applications. Florida: CRC Press; 1989. pp. 3–20. [Google Scholar]
  42. Mitsuhashi J., Goodwin R.H. The serum-free culture of insect cells in vitro. In: Mitsuhashi J, editor. Invertebrate Cell System Applications. Florida: CRC Press; 1989. pp. 31–43. [Google Scholar]
  43. Newland M., Greenfield P.F., Reid S. Hybridoma growth limitations: the roles of energy metabolism and ammonia production. Cytotechnology. 1990;3:215–229. doi: 10.1007/BF00365485. [DOI] [PubMed] [Google Scholar]
  44. Nielsen L.K., Smyth G.K., Greenfield P.F. Hemacytometer cell count distributions: implications of non-poisson behavior. Biotechnol. Prog. 1991;7:560–563. doi: 10.1021/bp00012a600. [DOI] [Google Scholar]
  45. Ohman L., Alarcon M., Ljunggren J., Ramqvist A.K., Haggstrom L. Glutamine is not an essential amino acid for Sf9 insect cells. Biotechnol. Lett. 1996;18:765–770. doi: 10.1007/BF00127885. [DOI] [Google Scholar]
  46. Popham H.J.R., Li Y., Miller L.K. Genetic improvement of Helicoverpa zea nuclear polyhedrosis virus as biopesticide. Biol. Control. 1997;10:83–91. doi: 10.1006/bcon.1997.0552. [DOI] [Google Scholar]
  47. Raghunard N., Dale B.E. Effects of glucose, glutamine and malate on the metabolism of Spodoptera frugiperda Clone 9 (Sf9) cells. An initial rate study. Appl. Biochem. Biotechnol. 1996;56:19–35. [Google Scholar]
  48. Reid S., Randerson D.H., Greenfield P.F. Amino acid determination in mammalian cell culture supernatants. Aust. J. Biotechnol. 1987;1:69–72. [Google Scholar]
  49. Rhiel M., Mitchell-Logean C.M., Murhammer D.W. Comparison of Trichoplusia ni BTI-Tn-5BI-4 (High Five) and Spodoptera frugiperda Sf9 insect cell line metabolism in suspension Cultures. Biotechnol. Bioeng. 1997;55:909–920. doi: 10.1002/(SICI)1097-0290(19970920)55:6<909::AID-BIT8>3.0.CO;2-K. [DOI] [PubMed] [Google Scholar]
  50. Rice W.C., McIntosh A.H., Ignoffo C.M. Yield and activity of the Heliothis zea single nuclear polyhedrosis virus propagated in cloned and uncloned lines of Heliothis cells. Vitro Cell. Dev. Biol. 1989;25:201–204. [Google Scholar]
  51. Rivkin H., Mor M., Chejanovsky N. Isolation, replication and polyhedrin gene sequence of an Israeli Helicoverpa armigera single nucleopolyhedrovirus. Virus Genes. 1998;17:11–19. doi: 10.1023/A:1008092631829. [DOI] [PubMed] [Google Scholar]
  52. Rosinski M. 2001. Modelling Insect Cell-Baculovirus Dynamics. PhD Thesis. The University of Queensland, p. 33.
  53. Ryll T., Valley U., Wagner R. Biochemistry of growth inhibition by ammonium ions in mammalian cells. Biotechnol. Bioeng. 1994;44:184–193. doi: 10.1002/bit.260440207. [DOI] [PubMed] [Google Scholar]
  54. Schlaeger E.J. Medium design for insect cell culture. Cytotechnology. 1996;20:57–70. doi: 10.1007/BF00350389. [DOI] [PubMed] [Google Scholar]
  55. Schlaeger E.J. The protein hydrolysate, Primatone RL, is a cost effective multiple growth promoter of mammalian cell culture in serum containing and serum free media and displays anti-apoptosis properties. J. Immunol. Methods. 1996;194:191–199. doi: 10.1016/0022-1759(96)00080-4. [DOI] [PubMed] [Google Scholar]
  56. Schneider M., Marison I.W., von Stockar U. The importance of ammonia in mammalian cell culture. J. Biotechnol. 1996;46:161–185. doi: 10.1016/0168-1656(95)00196-4. [DOI] [PubMed] [Google Scholar]
  57. Sugiura T., Amann E. Properties of two insect cell lines useful for the baculovirus expression system in serum-free culture. Biotechnol. Bioeng. 1996;51:494–499. doi: 10.1002/(SICI)1097-0290(19960820)51:4<494::AID-BIT13>3.0.CO;2-I. [DOI] [PubMed] [Google Scholar]
  58. Wang M.Y., Vakharia V., Bentley W.E. Expression of epoxide hydrolase in insect cells - a focus on the infected cell. Biotechnol. Bioeng. 1993;42:240–246. doi: 10.1002/bit.260420212. [DOI] [PubMed] [Google Scholar]
  59. Weiss S.A., Smith C.C., Vaughn J.L., Dougherty E.M., Tompkins G.J. Effect of aluminium chloride and zinc sulfate on Autographa californica nuclear polyhedrosis virus (AcNPV) replication in cell culture. In Vitro. 1982;18:937–944. doi: 10.1007/BF02796350. [DOI] [PubMed] [Google Scholar]
  60. Weiss S.A., Smith G.C., Kalter S.S., Vaughn J.L. Improved method for the production of insect cell cultures in large volume. In Vitro. 1981;17:495–502. [Google Scholar]
  61. Weiss S.A., Thomas D.W., Dunlop B.F., Georgis R., Vail P.V. and Hoffmann D.F. 1994. In vitro production of viral pesticides: key elements. In: Biopesticides: Opportunities for Australian Industry, Brisbane, Australia.
  62. Wong T.K.K., Nielsen L.K., Greenfield P.F., Reid S. Relationship between oxygen uptake rate and time of infection of Sf9 insect cells with a recombinant baculovirus. Cytotechnology. 1994;15:157–167. doi: 10.1007/BF00762390. [DOI] [PubMed] [Google Scholar]
  63. Yamada K., Sherman K.E., Maramorosch K. Serial passage of Heliothis zea singly embedded nuclear polyhedrosis virus in a homologous cell line. J. Invertebr. Pathol. 1982;39:185–191. doi: 10.1016/0022-2011(82)90008-8. [DOI] [Google Scholar]

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