, Volume 8, Issue 3, pp 237–248 | Cite as

Formation of bridges and large cellular clumps in CHO-cell microcarrier cultures: Effects of agitation dimethyl sulfoxide and calf serum

  • Michael C. Borys
  • Eleftherios T. Papoutsakis
Original Research Papers


We have investigated conditions that inhibit the tendency of CHO K1 cells to form cellular bridges between microcarriers and dense clumps of cellular overgrowth in microcarrier cultures. Microcarrier aggregation by cellular bridge formation was found to occur only during periods of rapid cell growth. The level of microcarrier aggregation decreased with increasing agitation intensity. Dense masses of cellular overgrowth formed inside bridges connecting the microcarriers and in clumps that protruded off the microcarrier surface. To replace cells that were continuously sheared from the microcarriers, cell growth occurred preferentially in areas of overgrowth after confluent microcarriers were maintained in a serum-free medium. This ultimately led to poor surface coverage as bare spots developed on the microcarrier away from the areas of dense cellular overgrowth. The development of bare spots was inhibited when confluent microcarriers were maintained in medium supplemented with 1% serum. The development of cellular overgrowth was inhibited by dimethyl sulfoxide. Thus, maintaining confluent microcarriers in medium supplemented with 1% dimethyl sulfoxide and 1% calf serum resulted in microcarriers that appeared similar to monolayer cultures. There was also a decrease in bridging in cultures supplemented with either 1% calf serum or 1% dimethyl sulfoxide/1% calf serum compared to serum-free cultures.

Key words

agitation bridging calf serum cell culture Chinese hamster ovary cells dimethyl sulfoxide microcarriers 


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  1. Avgerinos GC, Drapeau D, Socolow JS, Mao J, Hsiao K and Broeze RJ (1990) Spin filter perfusion system for high density cell culture: production of recombinant urinary type plasminogen activator in CHO cells. Bio/Technology 8: 54–58.CrossRefGoogle Scholar
  2. Bichay JT, Adams EG, Inch RW, Brewer JE and Bhuyan BK (1990) HPLC and flow cytometric analyses of uptake of adriamycin and menogaril by monolayers and multicell spheroids. Sel. Cancer Ther. 6: 153–166.Google Scholar
  3. Bliem R and Katinger H (1988) Scale-up engineering in animal cell technology: Part II. Trends Biotechnol. 6: 224–230.CrossRefGoogle Scholar
  4. Chapman AE and Calhoun JC (1988) Effects of glucose starvation and puromycin treatment on lipid-linked oligosaccharide precursors and biosynthetic enzymes in Chinese hamster ovary cells in vivo and in vitro. Arch. Biochem. Biophys. 260: 320–333.CrossRefGoogle Scholar
  5. Cherry RS and Papoutsakis ET (1986) Hydrodynamic effects on cells in agitated tissue culture reactors. Bioproc. Eng. 1: 29–41.CrossRefGoogle Scholar
  6. Cherry RS and Papoutsakis ET (1988) Physical mechanisms of cell damage in microcarrier cell culture bioreactors. Biotechnol. Bieng. 32: 1001–1014.CrossRefGoogle Scholar
  7. Cherry RS and Papoutsakis ET (1989) Modeling of contact-inhibited animal cell growth on flat surfaces and spheres. Biotechnol. Bioeng. 33: 300–305.CrossRefGoogle Scholar
  8. Clark JM and Hirtenstein MD (1981) Optimizing culture conditions for the production of animal cells in microcarrier culture. Ann. NY Acad. Sci. 369: 33–45.Google Scholar
  9. Colosi P, Talamantes F and Linzer DIH (1987) Molecular cloning and expression of mouse placental lactogen I complementary deoxyribonucleic acid. Molec. Endo. 1: 767–776.Google Scholar
  10. Dairkee SH and Glaser DA (1982) Dimethyl sulfoxide affects colony morphology on agar and alters distribution of glycosaminoglycans and fibronectin. Proc. Natl. Acad. Sci. USA 79: 6927–6931.CrossRefGoogle Scholar
  11. Elbien AD (1987) Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Ann. Rev. Biochem. 56: 497–534.CrossRefGoogle Scholar
  12. Friend C, Scher W, Holland JG and Sato T (1971) Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: stimulation of erythroid differentiation by dimethyl sulfoxide. Proc. Natl. Acad. Sci. USA 68: 378–382.Google Scholar
  13. Goetghebeur S and Hu WS (1991) Cultivation of anchorage-dependent animal cells in microsphere-induced aggregate culture. Appl. Microbiol. Biotechnol. 34: 735–741.CrossRefGoogle Scholar
  14. Hayter PM, Curling EMA, Baines AJ, Jenkins N, Salmon I, Strange PG, Tong JM and Bull AT (1991) Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant human interferon-γ. Biotechnol. Bioeng. In press.Google Scholar
  15. Horng C and McLimans W (1975) Primary suspension culture of calf anterior pituitary cells on a microcarrier surface. Biotechnol. Bioeng. 17: 713–732.CrossRefGoogle Scholar
  16. Ives CL, Eskin SG and McIntire LV (1986) Mechanical effects on endothelial cell morphology: in vitro assessment. In Vitro Cell. Develop. Biol. 22: 500–507.Google Scholar
  17. Kharasch N and Thyagarajan BS (1983) Structural basis for biological activities of dimethyl sulfoxide. Ann NY Acad. Sci. 411: 391–402.Google Scholar
  18. Kirsch AL, Kelley RO, Crissman H and Paxton L (1973) Dimethyl sulfoxide induced reversion of several features of polyoma transformed baby hamster kidney cells (BHK-21). J. Cell Biol. 37: 38–53.CrossRefGoogle Scholar
  19. Lampugnani MG, Pedenovi M, Niewiarowski A, Casali B, Donati MB, Corbascio GC and Marchisio PC (1987) Effects of dimethyl sulfoxide (DMSO) on microfilament organization, cellular adhesion and growth of cultured mouse B16 melanoma cells. Exp. Cell Res. 172: 385–396.CrossRefGoogle Scholar
  20. Layman DL (1987) Growth inhibitory effects of dimethyl sulfoxide and dimethyl sulfone on vascular smooth muscle and endothelial cell in vitro. In Vitro Cell. Dev. Biol. 23: 422–428.Google Scholar
  21. Levesque MJ, Sprague EA, Schwartz CJ and Nerem RM (1989) The influence of shear stress on cultured vascular endothelial cell: the stress response of an anchorage-dependent mammalian cell. Biotechnol. Prog. 5: 1–8.CrossRefGoogle Scholar
  22. Lin AA, Nguyen T and Miller WM (1991) A rapid method for counting cell nuclei using a particle sizer/counter. Biotech. Techniq. 5: 153–156.CrossRefGoogle Scholar
  23. Moore DS and McCabe GP (1989) Introduction to the Practice of Statistics (pp. 597–599). W. H. Freeman, New York.Google Scholar
  24. Ossowski L and Belin D (1985) Effect of dimethyl sulfoxide on human carcinoma cells, inhibition of plasminogen activator synthesis, change in cell morphology, and alteration of response to cholera toxin. Molec. Cell. Biol. 5: 3552–3559.Google Scholar
  25. Pharmacia Fine Chemicals Trade Publication (1981) Microcarrier cell culture. Principles and Methods, Upsala, Sweden.Google Scholar
  26. Ryan MP and Higgins PJ (1988) Cytomatrix reorganization in dimethyl sulfoxide ‘Qi’ substrate murine hepatic tumor cells. Int. J. Canc. 42: 273–278.Google Scholar
  27. Schulz R, Krafft H, Piehl GW and Lehmann J (1987) Production of human β-interferon in mouse L-cells. Develop. Biol. Standard. 66: 489–493.Google Scholar
  28. Stathopoulos NA and Hellums JD (1985) Shear stress effects on human embryonic kidney cells in vitro. Biotechnol. Bioeng. 27: 1021–1026.CrossRefGoogle Scholar
  29. Tanaka M, Levy J, Terada M, Breslow R, Rifkind RA and Marks PA (1975) Induction of erythroid differentiation in murine virus infected erythroleukemia cells by highly polar compounds. Proc. Natl. Acad. Sci. USA 72: 1003–1006.CrossRefGoogle Scholar
  30. Traganos F, Bueti C, Darzynkiewicz Z and Melamed MR (1984a) Effects of retinoic acid versus dimethyl sulfoxide on Friend erythroleukemia cell growth. I. Cell proliferation, RNA content and protein content. J. Natl. Canc. Inst. 73: 193–204.Google Scholar
  31. Traganos F, Higgins PJ, Bueti C, Darzynkiewicz Z and Melamed MR (1984b) Effects of retinoic acid versus dimethyl sulfoxide on Friend erythroleukemia cell growth. II. Induction of quiescent, non-proliferating cells. J. Natl. Canc. Inst. 73: 205–218.Google Scholar
  32. Varani J, Dame M, Veals TF and Wass JA (1983) Growth of three established cell lines on glass microcarriers. Biotechnol. Bioeng. 25: 1359–1372.CrossRefGoogle Scholar
  33. Wechezak AR, Viggers RF and Sauvage LR (1985) Fibronectin and F-actin redistribution in cultured endothelial cells exposed to shear stress. Lab. Invest. 53: 639–647.Google Scholar

Copyright information

© Kluwer Academic Publishers 1992

Authors and Affiliations

  • Michael C. Borys
    • 1
  • Eleftherios T. Papoutsakis
    • 1
  1. 1.Department of Chemical EngineeringNorthwestern UniversityEvanstonUSA

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