Cell Cycle Progression in Serum-Free Cultures of Sf9 Insect Cells: Modulation by Conditioned Medium Factors and Implications for Proliferation and Productivity (original) (raw)

Abstract

Cell cycle progression was studied in serum-free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum-free medium was characterized by an accumulation of 50-70% of the cells in the G 2 /M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G 1 and in particular into the S phase. Maximum rate of proliferation (µ N,max) was reached 24-48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G 2 /M phase. The following declining µ N could be explained by a slow increase in the G 2 /M cell population. However, at approximately 100 h, an abrupt increase in the amount of G 2 /M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G 2 /M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase µ N,max , decrease the time to reach µ N,max , and decrease the synchronization of cells in G 2 /M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of-galactosidase up to 68-75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean population cell volume had attained a minimum, and this occurred 24 h before the switch into the G 2 /M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G 2 /M cell cycle arrest.

Loading...

Loading Preview

Sorry, preview is currently unavailable. You can download the paper by clicking the button above.

References (63)

  1. References and Notes
  2. Cascio, M. Baculovirus expression of receptors and channels. In Methods in Neuroscience. Academic Press: New York, 1995; Vol. 25.
  3. Luckow, V. A.; Summers, M. D. Trends in the development of baculovirus expression vectors. Bio/Technology 1988, 6, 47-55.
  4. Lawrie, A. M.; King, L. A.; Ogden, J. E. High level synthesis and secretion of human urokinase using a late gene promoter of the Autographa californica nuclear polyhedrosis virus. J. Biotechnol. 1995, 39, 1-8.
  5. Brocks, B.; Rode, H. J.; Klein, M.; Gerlach, E.; Dubel, S.; Little, M.; Pfizermaier, K.; Moosmayer, D. A TNF receptor antagonistic scFv, which is not secreted in mammalian cells, is expressed as a soluble mono-and bivalent scFv derivative in insect cells. Immunotechnology 1997, 3, 173-184.
  6. Coleman, T. A.; Parmelee, D.; Thotakura, N. R.; Nguyen, N.; Bu ¨rgin, M.; Gentz, S.; Gentz, R. Production and purifica- tion of novel secreted human proteins. Gene 1997, 190, 163- 171.
  7. Volkman, L. E. Baculovirus bounty. Science 1995, 269, 1834.
  8. Doverskog, M.; Han, L.; Ha ¨ggstro ¨m, L. Cystine/cysteine metabolism in cultured Sf9 cells: Influence of cell physiology on biosynthesis, amino acid uptake and growth. Cytotechnol- ogy 1998, 26, 91-102.
  9. Radford, K. M.; Reid, S.; Greenfield, P. F. Substrate limita- tion in the baculovirus expression vector system. Biotechnol. Bioeng. 1997, 56, 32-44.
  10. Drews, M.; Paalme, T.; Vilu, R. The growth and nutrient utilization of the insect cell line Spodoptera frugiperda Sf9 in batch and continuous culture. J. Biotechnol. 1995, 40, 187- 198.
  11. Hensler, W.; Singh, V.; Agathos, S. N. Sf9 insect cell growth and β-galactosidase production in serum-free media. Ann. N.Y. Acad. Sci. 1994, 745, 149-168.
  12. Rhiel, M.; Mitchell-Logean, C. M.; Murhammer, D. W. Comparison of Trichoplusia ni BTI-Tn-5B1-4 (High Five) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures. Biotechnol. Bioeng. 1997, 55, 909-920.
  13. Bussolati, O.; Uggeri, J.; Rotoli, J.; Franchi-Gazzola, R.; Gazzola, G. C. The relationship between sodium dependent transport of anionic amino acids and cell proliferation. Biochim. Biophys. Acta 1993, 1151, 153-160.
  14. Bussolati, O.; Uggeri, J.; Belletti, S.; V., D.a.; Gazzola, G. C. The stimulation of Na, K, Cl cotransport and of system A for neutral amino acid transport is a mechanism for cell volume increase during the cell cycle. FASEB J. 1996, 10, 920-926.
  15. Collarini, E. J.; Oxender, D. L. Mechanisms of transport of amino acids across membranes. Ann. Rev. Nutr. 1987, 7, 75-90.
  16. Boonstra, J.; Mummery, C. L.; Tertoolen, L. G. J.; Van der Saag, P. T.; De Laat, S. W. Cation transport and growth regulation in neuroblastoma cells. Modulations of K + trans- port and electrical membrane properties during the cell cycle. J. Cell. Physiol. 1981, 107, 75-83.
  17. Wonderlin, W. F.; Strobl, J. S. Potassium channels, pro- liferation and G1 progression. J. Membr. Biol. 1996, 154, 91- 107.
  18. Be ´dard, C.; Kamen, A.; Tom, R.; Massie, B. Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures. Cytotechnology 1994, 15, 129-138.
  19. Lindsay, D. A.; Betenbaugh, M. J. Quantification of cell culture factors affecting recombinant protein yields in bacu- lovirus-infected insect cells. Biotechnol. Bioeng. 1992, 39, 614-618.
  20. Be ´dard, C.; Perret, S.; Kamen, A. A. Fed-batch culture of Sf9 cells supports 3 × 10 7 cells per mL and improves baculovirus-expressed recombinant protein yields. Biotechnol. Lett. 1997, 19, 629-632.
  21. Kioukia, N.; Nienow, A. W.; Emery, A. N.; Al-Rubeai, M. Physiological and environmental factors affecting the growth of insect cells and infection with baculovirus. J. Biotechnol. 1995, 38, 243-251.
  22. Zhang, J.; Kalogerakis, N.; Behie, L. A.; Iatrou, K. Opti- mum infection conditions for recombinant protein production in insect cell (Bm5) suspension culture. Biotechnol. Prog. 1994, 10, 636-643.
  23. Agathos, S. N. Insect cell bioreactors. Cytotechnology 1996, 20, 173-189.
  24. Brown, M.; Faulkner, P. Factors affecting the yield of virus in cloned cell line of Trichoplusia ni infected with Nuclear polyhedrosis virus. J. Inv. Pathol. 1975, 26, 251-257.
  25. Lynn, D. E.; Hink, W. F. Infection of synchronized TN- 368 cell cultures with Alfalfa looper nuclear polyhedrosis virus. J. Inv. Pathol. 1978, 32, 1-5.
  26. Fertig, G.; Klo ¨ppinger, M.; Miltenburger, H. G. Cell cycle kinetics of insect cell cultures compared to mammalian cell cultures. Exp. Cell Res. 1990, 189, 208-212.
  27. Vaughn, J. L.; Goodwin, R. H.; Tompkins, G. J.; McCawley, P. The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro 1977, 13, 213-217.
  28. Sawicki, W.; Kieler, J.; Briand, P. Vital staining with neutral red and trypan blue of 3H-thymidine-labeled cells prior to autoradiography. Stain Technol. 1967, 42, 143-146.
  29. Reuveny, S.; Kemp, C. W.; Eppstein, L.; Shiloach, J. Carbohydrate metabolism in insect cell cultures during cell growth and recombinant protein production. Ann. N.Y. Acad. Sci. 1992, 665, 230-237.
  30. Sangfelt, O.; Erickson, S.; Castro, J.; Heiden, T.; Einhorn, S.; Grande ´r, D. Induction of apoptosis and inhibition of cell growth are independent responses to interferon-R in hemato- poietic cell lines. Cell Growth Diff. 1997, 8, 343-352.
  31. Smith, A. V.; Orr-Weaver, T. L. The regulation of the cell cycle during Drosophila embryogenesis: the transition to polyteny. Development 1991, 112, 997-1008.
  32. Schopf, B.; Howaldt, M. W.; Baily, J. E. DNA distribution and respiratory activity of Spodoptera frugiperda population infected with wild-type and recombinant Autographa cali- fornica nuclear polyhedrosis virus. J. Biotechnol. 1990, 15, 169-186.
  33. Hillwig, I.; Eipel, H. E. Characterization of insect cell lines by DNA content. Z. Angew. Entomol. 1978/79, 87, 216-220.
  34. Lehner, C. F. Pulling the string: cell cycle regulation during Drosophila development. Cell Biol. 1991, 2, 223-231.
  35. Kylsten, P.; Saint, R. Imaginal tissues of Drosophila melanogaster exhibit different modes of cell proliferation control. Dev. Biol. 1997, 192, 509-522.
  36. Su, T. T.; Campbell, S. D.; O'Farrell, P. H. The cell cycle program in germ cells of the Drosophila embryo. Dev. Biol. 1998a, 196, 160-170.
  37. Broek, D.; Bartlett, R.; Crawford, K.; Nurse, P. Involvement of p34cdc2 in establishing the dependency of S phase on mitosis. Nature 1991, 349, 388-393.
  38. Sennerstam, R.; Stro ¨mberg, J.-O. Cell cycle progression: computer simulation of uncoupled subcycles of DNA replica- tion and cell growth. J. Theor. Biol. 1995, 175, 177-189.
  39. Knoblich, J. A.; Sauer, K.; Jones, L.; Richardson, H.; Saint, R. Cyclin E controls S phase progression and its down- regulation during Drosophila emryogenesis is required for the arrest of cell proliferation. Cell 1994, 77, 107-120.
  40. Hammond, M. P.; Laird, C. D. Chromosome structure and DNA replication in nurse and follicle cells of Drosophila melanogaster. Chromosoma 1985, 91, 267-278.
  41. Braunagel, S. C.; Parr, R.; Belyavskyi, M.; Summers, M. D. Autographa californica nucleopolyhedrovirus infection results in Sf9 cell cycle arrest at G2/M phase. Virology 1998, 244, 195-211.
  42. Ikeda, M.; Kobayashi, M. Cell-cycle perturbation in Sf9 cells infected with Autographa californica nucleopolyhedrovi- rus. Virology 1999, 258, 176-188.
  43. Jesionowski, G. A.; Ataai, M. M. An efficient medium for high protein production in the insect cell/baculovirus expres- sion system. Biotechnol. Prog. 1997, 13, 355-360.
  44. Lauffenburger, D.; Cozens, C. Regulation of mammalian cell growth by autocrine growth factors: analysis of conse- quences for inoculum cell density effects. Biotechnol. Bioeng. 1989, 33, 1365-1378.
  45. Rubin, H. A substance in conditioned medium which enhances the growth of small numbers of chick embryo cells. Exp. Cell res. 1966, 41, 138-148.
  46. Lee, G. M.; Kaminski, M. S.; Palsson, B. O. Observations consistent with autocrine stimulation of hybridoma cell growth and implications for large-scale antibody production. Biotechnol. Lett. 1992, 14, 257-262.
  47. Huang, L. C.; Wu, J. H. D. Cell density-dependent prolif- eration of the murine bone marrow-derived stromal cell lines. Biotechnol. Lett. 1997, 19, 89-92.
  48. Lee, C. W. T.; Shuler, M. L. The effect of inoculum density and conditioned medium on the production of Ajmalicine and Catharanthine from immobilized Catharanthus roseus cells. Biotechnol. Bioeng. 1999, 67, 61-71.
  49. Wu, J.; Ruan, Q.; Lam, H. Y. P. Evaluation of spent medium recycle and nutrient feeding strategies for recombi- nant protein production in the insect cell-baculovirus process. J. Biotechnol. 1998, 66, 109-116.
  50. Neutra, R.; Levi, B.-Z.; Shoham, Y. Optimization of protein- production by the baculovirus expression vector system in shake flasks. Appl. Microbiol. Biotechnol. 1992, 37, 74-78.
  51. Lazarte, J. E.; Tosi, P.-F.; Nicolau, C. Optimization of the production of full length rCD4 in baculovirus infected Sf9 cells. Biotechnol. Bioeng. 1992, 40, 214-217.
  52. Hayakawa, Y.; Ohnishi, A. Cell growth activity of growth- blocking peptide. Biochem. Biophys. Res. Comm. 1998, 250, 194-199.
  53. Doverskog, M.; Tally, M.; Ha ¨ggstro ¨m, L. 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. 1999, 265, 674-679.
  54. Schlaeger, E.-J. Medium design for insect cell culture. Cytotechnology 1996, 20, 57-70.
  55. Hensler, W. T.; Agathos, S. N. Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus infected insect cells. Cyto- technology 1994, 15, 177-186.
  56. Elias, C. B.; Zeiser, A.; Be ´dard, C.; Kamen, A. A. Enhanced growth of Sf-9 cells to a maximum density of 5.2 × 10 7 cells per mL and production of β-galactosidase at high cell density by fed batch culture. Biotechnol. Bioeng. 2000, 68, 381-388.
  57. Nguyen, B.; Jarnagin, K.; Williams, S.; Chan, H.; Barnett, J. 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. 1993, 31, 205-217.
  58. Rhoads, J. M.; Argenzio, R. A.; Chen, W.; Graves, L. M.; Licato, L. L.; Blikslager, A. T.; Smith, J.; Gatzy, J.; Brenner, D. A. Glutamine metabolism stimulates intestinal cell MAPKs by a cAMP-inhibitable Raf-independent mechanism. Gastro- enterology 2000, 118, 90-100.
  59. Garrington, T. P.; Johnson, G. L. Organization and regula- tion of mitogen-activated protein kinase signaling pathways. Curr. Opin. Cell. Biol. 1999, 11, 211-218.
  60. Itoh, T.; Yamauchi, A.; Miyai, A.; Yokoyama, K.; Kamada, T.; Ueda, N.; Fujiwara, Y. Mitogen-activated protein kinase and its activator are regulated by hypertonic stress in Madin- Darby canine cells. J. Clin. Invest. 1994, 72, 2387-2390.
  61. Dall'Asta, V.; Rossi, P. A.; Bussolati, O.; Gazzola, G. C. Response of human fibroblasts to hypertonic stress. Cell shrinkage is counteracted by an enhanced active transport of neutral amino acids. J. Biol. Chem. 1994, 8, 10485-10491.
  62. Drews, M.; Kasemets, K.; Nisamedtinov, I.: Paalme, T. Continuous cultivation of insect and yeast cells at maximum specific growth rate. Proc. Estonian Acad. Sci. Chem. 1998, 47, 175-188.
  63. Zeng, A.-P.; Deckwer, W.-D.; Hu, W.-S. Determinants and rate laws of growth and death of hybridoma cells in continu- ous culture. Biotechnol. Bioeng. 1998, 57, 642-654.