Biotechnology and Bioprocess Engineering

, Volume 3, Issue 2, pp 87–90 | Cite as

Oxidation-deficient silkworm hemolymph as a medium supplement for insect cell culture

  • Eun Jeong Kim
  • Ji-Young Choi
  • Sam-Eun Kim
  • Tai Hyun Park


Hemolymph is oxidized and darkens visibly during the collection from silkworms due to the activity of tyrosinase in it. Toxic quinones are produced by the oxidation and consequently inhibit the cell growth. Heat treatment can be used to prevent the oxidation; however, the oxidation may occur during the collection of hemolymph before it is heat-treated. It makes the hemolymph collection difficult especially on a large-scale preparation. Hemolymphs collected from 257 different strains of silkworms were examined to select the slowly oxidized hemolymphs. Hemolymphs collected from mutant strains such as Lemone, TBO, Cre, Y4, and wEb showed relatively slow color changes. Oxidation rates of the hemolymphs were measured by the absorbance change using a spectrophotometer. The hemolymph of wEb showed the slowest oxidation. The absorbance of this mutant hemolymph reached the saturation value at 20°C in 450 min, whereas the total oxidation time of the wild-type (Baekokjam) hemolymph at the same temperature was 120 min. We tested if this mutant hemolymph is useful as a medium supplement for insect cell culture. Cell growth rate and final cell concentration in the medium supplemented with the wEb hemolymph were almost same as those in the medium supplemented with the wild-type hemolymph. Hemolymph is collected on a small scale by clipping the abdominal leg; however, this method is not appropriate for large scale preparation. Centrifugation after chopping the silkworm hemolymph by a blending mixer is a more appropriate procedure for large scale collection. Slowly oxidized wEb hemolymph resulted in higher cell concentration than the wild-type hemolymph when hemolymph was collected by the large scale preparation method.

Key words

silkworm hemolymph insect cell oxidation medium supplement 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. [1]
    Miltenburguer, H. G. (1982) Investigation on the cultivation of insect cell lines in serum free media. p. 31–43. In: G. Fisher and R J. Wieser (ed.)Hormonally defined media: A tool in cell biology, Springer-Verlag, New York, NY.Google Scholar
  2. [2]
    Wyatt, G. R., T. C. Lougheed, and S. S. Wyatt (1956) The chemistry of insect hemolymph-organic components of the hemolymph of the silkworm,Bombyx mori, and two other species.J. Gen. Physiol. 39: 853–868.CrossRefGoogle Scholar
  3. [3]
    Wyatt, S. S. (1956) Culture in vitro of tissue from the silkwormBombyx mori L.J. Gen. Physiol. 39: 841–852.CrossRefGoogle Scholar
  4. [4]
    Grace, T. D. C. (1962) Establishment of four strains of cells from insect tissue grownin vitro.Nature 195: 788–789.CrossRefGoogle Scholar
  5. [5]
    Zhang, J., N. Kalogeraskis, L. Behie, and K. Iatou (1992) Investigation of reduced serum and serum-free media for cultivation of insect cells (Bm5) and the production of baculovirus (BmNPV).Biotechnol. Bioeng. 40: 1165–1172.CrossRefGoogle Scholar
  6. [6]
    Ha, S. H., T. H. Park, and S.-E. Kim (1996) Silkworm hemolymph as a substitute for fetal bovine serum in insect cell culture.Biotechnol. Techniques 10: 401–406.CrossRefGoogle Scholar
  7. [7]
    Ha, S. H. and T. H. Park (1997) Efficient production of recombinant protein inSpodoptera frugiperda/AcNPV system utilizing silkworm hemolymph.Biotechnol. Lett. 19: 1087–1091.CrossRefGoogle Scholar
  8. [8]
    Summers, M. D. and G. E. Smith (1987) A mannual of method for baculovirus vectors and insect cell culture procedures.Tex. Agric. Exp. Stn. Bull. 1555: 1–56.Google Scholar
  9. [9]
    Raper, H. H. (1928) The anaerobic oxidase.Physiol. Rev. 8: 245–282.Google Scholar
  10. [10]
    Mason, H. S. (1948) The chemistry of melanin III mechanism of the oxidation of dihydroxyphenylalanine by tyrosinase.J. Biol. Chem. 172: 83–99.Google Scholar
  11. [11]
    Sugumaran, M. and V. Semensi (1991) Quinone methide as a new intermediate in eumelanin biosynthesis.J. Biol. Chem. 266(10): 6073–6078.Google Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering 1998

Authors and Affiliations

  • Eun Jeong Kim
    • 1
  • Ji-Young Choi
    • 2
  • Sam-Eun Kim
    • 2
  • Tai Hyun Park
    • 1
  1. 1.School of Chemical EngineeringSeoul National UniversitySeoulKorea
  2. 2.Department of Sericulture and EntomologyNational Institute of Agricultural Science and TechnologySuwonKorea

Personalised recommendations