Skip to main content
Log in

Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultues

  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

Temperature reduction in CHO cell batch culture may be beneficial in the production of recombinant protein and in maintenance of viability. The effects on cell cycle, apoptosis and nucleotide pools were studied in cultures initiated at 37°C and temperature shifted to 30 °C after 48 hours. In control cultures maintained at 37 °C, viable cells continued to proliferate until the termination of the culture, however, temperature reduction caused a rapid decrease in the percent of cells in S phase and accumulation of cells in G-1. This was accompanied by a concurrent reduction in U ratio (UTO/UDP-GNAc), previously shown to be a sensitive indicator of growth rate. Culture viability was extended following temperature shift, as a result of delayed onset of apoptosis, however, once initiated, the rate and manner of cell death was similar to that observed at 37 °C. All nucleotide pools were similarly degraded at the time of apoptotic cell death. Temperature reduction to 30 °C did not decrease the energy charge of the cells, however, the overall rate of metabolism was reduced. The latter may be sufficient to extend culture viability via a reduction in toxic metabolites and/or limitation of nutrient deprivation. However, the possibility remains that the benefits of temperature reduction in terms of both viability and productivity are more directly associated with cultures spending extended time in G-1.

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

  • Barnabé N and Butler M (1994) Effect of temperature on nucleotide pools and monoclonal antibody production in a mouse hybridoma. Biotechnology and Bioengineering, 44: 1235–1245.

    Google Scholar 

  • Bloemkolk JW, Gray MR, Merchant F and Mosmann TR (1992) Effect of temperature on hybridoma cell cycle and MAb production. Biotechnology and Bioengineering 40: 427–431.

    Google Scholar 

  • de la Broise D, Noiseux M, Lemieux R and Massie B (1991) Long term perfusion culture of hybridoma: a 'grow or die' cell cycle system. Biotechnology and Bioengineering 38: 781–787.

    Google Scholar 

  • Coco-Martin Jm, Oberink JW, van der Velden-de Groot TAM and Beuvery EC (1992) The potential of flow cytometric analysis for the characterization of hybridoma cells in suspension cultures. Cytotechnology 8: 65–74.

    Google Scholar 

  • Dean PN and Jett JH (1974) Mathematical analysis of DNA distribution derived from flow microfluorimetry. J. Cell Biol. 60: 523–527.

    Google Scholar 

  • Fraňek F, Vomastek T and Dolníková J (1992) Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures. Cytotechnology 9: 117–123.

    Google Scholar 

  • Giard DJ, Fleischaker RJ and Fabrikant M (1982) Effect of temperature on the production of human fibroblast interferon (41411). Proc Soc Exp Biol Med 170: 155–159.

    Google Scholar 

  • Goergen JL, Marc A and Engasser JM (1993) Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release. Cytotechnology 11: 189–195.

    Google Scholar 

  • Gorczyca W, Bigman K, Mittleman A, Ahmed T, Gong J, Melamed MR and Darzynkiewicz Z (1993) Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia 7: 659–670.

    Google Scholar 

  • Gorczyca W, Bruno S, Darzynkiewicz RJ, Gong J and Darzynkiewicz Z (1992) DNA strand breaks occurring during apoptosis: their early in situ detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. International Journal of Oncology 1: 639–648.

    Google Scholar 

  • Jenkins N and Hovey A (1993) Temperature control of growth and productivity in mutant Chinese hamster ovary cells synthesizing a recombinant protein. Biotechnology and Bioengineering 42: 1029–1036.

    Google Scholar 

  • Kaufman RJ and Sharp PA (1982) Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene. J Mol Biol 159: 601–621.

    Google Scholar 

  • Moore A, Donahue CJ, Hooley J, Stocks DL, Bauer KD and Mather JP (1995) Apoptosis in CHO cell batch cultures: examination by flow cytometry. Cytotechnology 17: 1–11.

    Google Scholar 

  • Müller M, Siems W, Buttgereit F, Dumdey and Rapoport SM (1986) Quantification of ATP-producing and consuming processes of Ehrlich ascites tumor cells. Eur J Biochem 161: 701–705.

    Google Scholar 

  • Ramírez OT and Mutharasan R (1990) Cell cycle-and growth phase — dependent variations in size distribution, antibody productivity and oxygen demand in hybridoma cultures. Biotechnol Bioeng 36: 839–848.

    Google Scholar 

  • Reuveny S, Velez D, Maxmillan JD and Miller L (1986) Factors affecting cell growth and monoclonal antibody production in stirred reactors. J Immunol Methods 86: 53–59.

    Google Scholar 

  • Reuveny S, Kim YJ, Kemp CW and Shiloach J (1993) Effect of temperature and oxygen on cell growth and recombinant protein production in insect cell cultures. Appl. Microbiol Biotechnol 38: 619–623.

    Google Scholar 

  • Ryll T and Wagner R (1991) Improved ion-pair high-performance liquid chromatographic method for the quantification of a wide variety of nucleotides and sugar-nucleotides in animal cells. Journal of Chromatography, 570: 77–88.

    Google Scholar 

  • Ryll T and Wagner R (1992) Intracellular ribonucleotide pools as a tool for monitoring the physiological state of in vitro cultivated mammalian cells during production processes. Biotechnology and Bioengineering, 40: 934–946.

    Google Scholar 

  • Ryll T, Valley U and Wagner R (1994) Biochemistry of growth inhibition by ammonium ions in mammalian cells. Biotechnology and Bioengineering, 44: 184–193.

    Google Scholar 

  • Schwartzman RA, Cidlowski JA (1993) Mechanism of tissue-specific induction of internucleosomal deoxyribonucleic acid cleavage activity and apoptosis by glucocorticoids. Endocrinology 133: 591–599.

    Google Scholar 

  • Singh RP, Al-Rubeai M, Gregory CD and Emery AN (1994a) Cell death in bioreactors: a role for apoptosis. Biotechnol Bioeng 44: 720–726.

    Google Scholar 

  • Singh M, Al-Rubeai M, Goldman MH and Emery AN (1994b) Apoptosis — the programme for cell death in agitated bioreactors. Biotechnol 94ABE2, 23–25.

    Google Scholar 

  • Sureshkumar GK and Muthaeasan R (1991) The influence of temperature on a mouse-mouse hybridoma growth and monoclonal antibody production Biotechnol Bioeng 37: 292–295.

    Google Scholar 

  • Suzuki E and Ollis DF (1989) Cell cycle model for antibody production kinetics Biotechn Bioeng 34: 1398.

    Google Scholar 

  • Telford WG, King LE and Fraker PJ (1991) Evaluation of glucocorticoid-induced DNA fragmentation in mouse thymocytes by flow cytometry. Cell Prolif 24: 447–459.

    Google Scholar 

  • Tilly JL and Hsueh AJW (1993) Microscale autoradiographic method for the quanlitative and quantitative analysis of apoptotic DNA fragmentation. J Cell Physiol 154: 519–626.

    Google Scholar 

  • Urlaub G and Chasin LA (1980) Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci USA 77: 4216–4220.

    Google Scholar 

  • Weidemann R, Ludwig A and Kretzmer G (1994) Low temperature cultivation — A step towards process optimization. Cytotechnology 15: 111–116.

    Google Scholar 

  • Zaharevitz DA, Napier EA, Anderson LW, Strong JM and Cysyk RL (1988) Stimulation of uracil nucleotide synthesis in mouse liver, intestine and kidney by ammonium chloride infusion. European Journal of Biochemistry, 175: 193–198.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Moore, A., Mercer, J., Dutina, G. et al. Effects of temperature shift on cell cycle, apoptosis and nucleotide pools in CHO cell batch cultues. Cytotechnology 23, 47–54 (1997). https://doi.org/10.1023/A:1007919921991

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1007919921991

Navigation