Skip to main content
Log in

Effect of Partial Medium Replacement on Cell Growth and Protein Production for the High-Five™ insect cell line

  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

The potential of spent medium to support the growth and recombinant protein production of High-Five™ cells was investigated. Growth in medium consisting of three parts fresh and one part spent medium was comparable to that in fresh medium (maximal specific growth rates of 0.028 and 0.029 h−1, and maximal cell densities of 4 and 4.5 × 106 cells ml−1, respectively). Glucose exhaustion coincided with an abrupt decrease of viability. Of 15 amino acids analyzed, not a single one was completely exhausted at the end of the growth phase. Growth in medium consisting of equal parts spent and fresh medium led to lower maximal cell concentration (2.9 × 106 cells ml−1) with a smoother death phase. Glucose supplementation at the beginning of the culture or at the end of the growth phase did not lead to an increase of either the maximal cell density or the specific growth rate. Infection of High-Five™ cells at three different densities (1.4, 2.5 and 4.2 × 106 cells ml−1) without medium change led to monotonically decreased specific productions for β-galactosidase. Partial (75%) or total medium replacement at the higher infection density restored the specific production at the levels of the intermediate density infection (321, 292 and 389 U.(106 cells)−1, respectively).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Agathos S.N. 1996. Insect cell bioreactors. Cytotechnology 20: 173–189.

    Google Scholar 

  • Agathos S.N., Jeong Y.-H. & Venkat K. 1990. Growth kinetics of free and immobilized insect cell cultures. Ann NY Acad Sci 589: 372–398.

    Google Scholar 

  • Bédard C., Jolicoeur M., Jardin B., Tom R., Perret S. & Kamen A. 1994a. Insect cell density in bioreactor cultures can be estimated from on-line measurements of optical density. Biotechnol Tech 8: 605–610.

    Google Scholar 

  • Bédard C., Kamen A., Tom R.& Massie B. 1994b. Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures. Cytotechnology 15: 129–138.

    Google Scholar 

  • Bédard C., Perret S. & Kamen A.A. 1997. Fed-batch culture of Sf-9 cells supports 3 x 107 cells per ml and improves baculovirus-expressed recombinant protein yields. Biotechnol Lett 19: 629–632.

    Google Scholar 

  • Bédard C., Tom R. & Kamen A. 1993. Growth, nutrient consumption, and end-product accumulation in Sf-9 and BTIEAA insect cell cultures: Insights into growth limitation and metabolism. Biotechnol Prog 9: 615–624.

    Google Scholar 

  • Chico E. & Jäger V. 2000. Perfusion culture of baculovirus-infected BTI-Tn-5B1-4 insect cells: A method to restore cell-specific β-trace glycoprotein productivity at high cell density. Biotechnol Bioeng 70: 574–586.

    Google Scholar 

  • Dee K.U., Shuler M.L. & Wood H.A. 1997. Inducing single-cell suspension of BTI-TN5B1-4 insect cells: I. The use of sulfated polyanions to prevent cell aggregation and enhance recombinant protein production. Biotechnol Bioeng 54: 191–205.

    Google Scholar 

  • Donaldson M., Wood H.A., Kulakosky P.C. & Shuler M.L. 1999. Glycosylation of a recombinant protein in the Tn5B1-4 insect cell line: Influence of ammonia, time of harvest, temperature, and dissolved oxygen. Biotechnol Bioeng 63: 255–262.

    Google Scholar 

  • Doverskog M., Bertram E., Ljunggren J., Öhman L., Sennerstam R. & Häggström L. 2000. Cell cycle progression in serum-free cultures of Sf9 insect cells: modulation by conditioned medium factors and implications for proliferation and productivity. Biotechnol Prog 16: 837–846.

    Google Scholar 

  • Doverskog M., Ljunggren J., Öhman L. & Häggström L. 1997. Physiology of cultured animal cells. J Biotechnol 59: 103–115.

    Google Scholar 

  • Doverskog M., Tally M. & Häggström L. 1999. Constitutive secretion of an endogenous insulin-like peptide binding protein with high affinity for insulin in Spodoptera frugiperda (Sf9) cell cultures. Biochem Biophys Res Commun 265: 674–679.

    Google Scholar 

  • Drews M., Paalme T. & Vilu R. 1995. The growth and nutrient utilization of the insect cell line Spodoptera frugiperda Sf9 in batch and continuous culture. J Biotechnol 40: 187–198.

    Google Scholar 

  • Elias C.B., Zeiser A., Bédard C. & Kamen A.A. 2000. Enhanced growth of Sf-9 cells to a maximum density of 5.2β107 cells per ml and production of β-galactosidase at high cell density by fed batch culture. Biotechnol Bioeng 68: 381–388.

    Google Scholar 

  • Fekkes D., van Dalen A., Edelman M. & Voskuilen A. 1995. Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde. J Chromatogr B 669: 177–186.

    Google Scholar 

  • Ferkovich S.M. & Oberlander H. 1991. Growth factors in invertebrate in vitro culture. In Vitro Cell Dev Biol 27: 483–486.

    Google Scholar 

  • Ferrance J.P., Goel A. & Ataai M.M. 1993. Utilization of glucose and amino acids in insect cells cultures: Quantifying the metabolic flows within the primary pathways and medium development. Biotechnol Bioeng 42: 697–707.

    Google Scholar 

  • Gilbert R.S., Nagano Y., Yokota T., Hwan S.F., Fletcher T. & Lydersen K. 1996. Effect of lipids on insect cell growth and expression of recombinant proteins in serum-free medium. Cytotechnology 22: 211–216.

    Google Scholar 

  • Hensler W., Singh V. & Agathos S.N. 1994. Sf9 insect cell growth and β-galactosidase production in serum and serum-free media. Ann NY Acad Sci 745: 149–166.

    Google Scholar 

  • Ikonomou L., Bastin G., Schneider Y.J. & Agathos S.N. 2001. Design of an efficient medium for insect cell growth and recombinant protein production. In Vitro Cell Dev Biol-Anim 37: 549–559.

    Google Scholar 

  • Ikonomou L., Drugmand J.C., Bastin G., Schneider Y.J. & Agathos S.N. 2002. Microcarrier culture of lepidopteran cell lines: Implications for growth and recombinant protein production. Biotechnol Prog 18: 1345–1355.

    Google Scholar 

  • Ikonomou L., Schneider Y.J. & Agathos S.N. 2003. Insect cell culture for industrial production of recombinant proteins. Appl Microbiol Biotechnol 62: 1–20.

    Google Scholar 

  • Jarvis D.L. 2003. Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production. Virology 310: 1–7.

    Google Scholar 

  • Jesionowski G.A. & Ataai M.M. 1997. An efficient medium for high protein production in the insect cell/baculovirus expression system. Biotechnol Prog 13: 355–360.

    Google Scholar 

  • JRH Biosciences 2000. EX-CELLTM serum-free media: The 400 Series.

  • Kempken R., Büntemeyer H. & Lehmann J. 1991. The medium cycle bioreactor (MCB): Monoclonal antibody production in a new economic production system. Cytotechnology 7: 63–74.

    Google Scholar 

  • Kioukia N., Al-Rubeai M., Zhang Z., Emery A.N., Nienow A.W. & Thomas C.R. 1995a. A study of uninfected and baculovirus infected Spodoptera frugiperda cells in T-and spinner flasks. Biotechnol Lett 17: 7–12.

    Google Scholar 

  • Kioukia N., Nienow A.W., Al-Rubeai M. & Emery A.N. 1996. Influence of agitation and sparging on the growth rate and infection of insect cells in bioreactors and a comparison with hybridoma cultures. Biotechnol Prog 12: 779–785.

    Google Scholar 

  • Kioukia N., Nienow A.W., Emery A.N.& Al-Rubeai M. 1995b. Physiological and environmental factors affecting the growth of insect cells and infection with baculovirus. J Biotechnol 38: 243–251.

    Google Scholar 

  • Lauffenburger D. & Cozens C. 1989. Regulation of mammalian cell growth by autocrine growth factors: Analysis of consequences for inoculum cell density effects. Biotechnol Bioeng 33: 1365–1378.

    Google Scholar 

  • Lee G.M., Kaminski M.S. & Palsson B.O. 1992. Observations consistent with autocrine stimulation of hybridoma cell growth and implications for large-scale antibody production. Biotechnol Lett 14: 257–262.

    Google Scholar 

  • Lindsay D.A. & Betenbaugh M.J. 1992. Quantification of cell culture factors affecting recombinant protein yields in baculovirus-infected insect cells. Biotechnol Bioeng 39: 614–618.

    Google Scholar 

  • Luckow V.A. 1991. Cloning and expression of heterologous genes in insect cells with baculovirus vectors.In:Prokop A. & Bajpai P. (eds), Recombinant DNA Technology and Application, McGraw Hill, New York, pp. 97–152.

    Google Scholar 

  • Melkonyan H.S., Chang W.C., Shapiro J.P., Mahadevappa M., Fitzpatrick P.A., Kiefer M.C., Tomei L.D. & Umansky S.R. 1997. SARPs: A family of secreted apoptosis-related proteins. Proc Natl Acad Sci USA 94: 13636–13641.

    Google Scholar 

  • Mendonca R.Z., Palomares L.A. & Ramirez O.T. 1999. An insight into insect cell metabolism through selective nutrient manipulation. J Biotechnol 72: 61–75.

    Google Scholar 

  • Miller J.H. 1972. Assay of β-galactosidase. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, New York, pp.352–355.

    Google Scholar 

  • Nguyen B., Jarnagin K., Williams S., Chan H. & Barnett J. 1993. Fed-batch culture of insect cells: A method to increase the yield of recombinant human nerve growth factor (rhNGF) in the baculovirus expression system. J Biotechnol 31: 205–217.

    Google Scholar 

  • Nishino H. & Mitsuhashi J. 1995. Effects of some mammalian growth promoting substances on insect cell cultures. In Vitro Cell Dev Biol 31: 822–823.

    Google Scholar 

  • Öhman L., Ljunggren J. & Häggström L. 1995. Induction of a metabolic switch in insect cells by substrate-limited fed batch cultures. Appl Microbiol Biotechnol 43: 1006–1013.

    Google Scholar 

  • Polazzi E., Gianni T. & Contestabile A. 2001. Microglial cells protect cerebellar granule neurons from apoptosis: Evidence for reciprocal signaling. Glia 36: 271–280.

    Google Scholar 

  • Radford K.M., Reid S. & Greenfield P.F. 1997. Substrate limitation in the baculovirus expression vector system. Biotechnol Bioeng 56: 32–44.

    Google Scholar 

  • Reuveny S., Kim Y.J., Kemp C.W. & Shiloach J. 1993. Production of recombinant proteins in high-density insect cell cultures. Biotechnol Bioeng 42: 235–239.

    Google Scholar 

  • Rhiel M., Mitchell-Logean C.M. & Murhammer D.W. 1997. Comparison of Trichoplusia ni BTI-Tn-5B1-4 (High FiveTM) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures. Biotechnol Bioeng 55: 909–920.

    Google Scholar 

  • Riese U., Lutkemeyer D., Heidemann R., Büntemeyer H. & Lehmann J. 1994. Reuse of spent cell culture medium in pilot-scale and rapid preparative purification with membrane chromatography. J Biotechnol 34: 247–257.

    Google Scholar 

  • Roberts P.L. 1984. Growth of insect cells in recycled medium and the use of various serum supplements. Biotechnol Lett 6: 633–638.

    Google Scholar 

  • Schlaeger E.-J. 1996. The protein hydrolysate, Primatone R.L., 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 194: 191–199.

    Google Scholar 

  • Taticek R.A., Choi C., Phan S.E., Palomares L.A. & Shuler M.L. 2001. Comparison of growth and recombinant protein production in two different insect cell lines in attached and suspension culture. Biotechnol Prog 17: 676–684.

    Google Scholar 

  • Taticek R.A. & Shuler M.L. 1997. Effect of elevated oxygen and glutamine levels on foreign protein production at high cell densities using the insect cell-baculovirus expression system. Biotechnol Bioeng 54: 142–152.

    Google Scholar 

  • Toku K., Tanaka J., Yano H., Desaki J., Zhang B., Yang L.H., Ishihara K., Sakanaka M. & Maeda N. 1998. Microglial cells prevent nitric oxide-induced neuronal apoptosis in vitro. J Neurosci Res 53: 415–425.

    Google Scholar 

  • Tom R.L., Debanne M.T., Bédard C., Caron A.W., Massie B. & Kamen A.A. 1995. Improved yields of the extracellular domain of the epidermal growth factor receptor produced using the baculovirus expression system by medium replacement following infection. Appl Microbiol Biotechnol 44: 53–58.

    Google Scholar 

  • Wang M.-Y., Kwong S. & Bentley W.E. 1993. Effects of oxygen/ glucose/glutamine feeding on insect cell baculovirus protein expression: A study on epoxide hydrolase production. Biotechnol Prog 9: 355–361.

    Google Scholar 

  • Wu J.-Y., Ruan Q. & Lam H.Y.P. 1998. Evaluation of spent medium recycle and nutrient feeding strategies for recombinant protein production in the insect cell-baculovirus process. J Biotechnol 66: 109–116.

    Google Scholar 

  • Yang J.-D., Gecik P., Collins A., Czarnecki S., Hsu H.-H., Lasdun A., Sundaram R., Muthukumar G.& Silberklang M. 1996. Rational scale-up of a baculovirus-insect batch process based on medium nutritional depth. Biotechnol Bioeng 52: 696–706.

    Google Scholar 

Download references

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ikonomou, L., Bastin, G., Schneider, YJ. et al. Effect of Partial Medium Replacement on Cell Growth and Protein Production for the High-Five™ insect cell line. Cytotechnology 44, 67–76 (2004). https://doi.org/10.1023/B:CYTO.0000043413.53044.fa

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/B:CYTO.0000043413.53044.fa

Navigation