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. 1997 Jan;23(1-3):47–54. doi: 10.1023/A:1007919921991

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

Alison Moore 1, Jennifer Mercer 1, George Dutina 2, Christopher J Donahue 3, Kenneth D Bauer 3, Jennie P Mather 1, Tina Etcheverry 2, Thomas Ryll 2,✉
PMCID: PMC3449885  PMID: 22358520

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.

Keywords: apoptosis, programmed cell death, nucleotides, energy charge, CHO cells, batch culture

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References

  1. Barnabé N, Butler M. Effect of temperature on nucleotide pools and monoclonal antibody production in a mouse hybridoma. Biotechnology and Bioengineering. 1994;44:1235–1245. doi: 10.1002/bit.260441011. [DOI] [PubMed] [Google Scholar]
  2. Bloemkolk JW, Gray MR, Merchant F, Mosmann TR. Effect of temperature on hybridoma cell cycle and MAb production. Biotechnology and Bioengineering. 1992;40:427–431. doi: 10.1002/bit.260400312. [DOI] [PubMed] [Google Scholar]
  3. de la Broise D, Noiseux M, Lemieux R, Massie B. Long term perfusion culture of hybridoma: a 'grow or die' cell cycle system. Biotechnology and Bioengineering. 1991;38:781–787. doi: 10.1002/bit.260380712. [DOI] [PubMed] [Google Scholar]
  4. Coco-Martin J., Oberink JW, van der Velden-de Groot TAM, Beuvery EC. The potential of flow cytometric analysis for the characterization of hybridoma cells in suspension cultures. Cytotechnology. 1992;8:65–74. doi: 10.1007/BF02540031. [DOI] [PubMed] [Google Scholar]
  5. Dean PN, Jett JH. Mathematical analysis of DNA distribution derived from flow microfluorimetry. J. Cell Biol. 1974;60:523–527. doi: 10.1083/jcb.60.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fraňek F, Vomastek T, Dolníková J. Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures. Cytotechnology. 1992;9:117–123. doi: 10.1007/BF02521738. [DOI] [PubMed] [Google Scholar]
  7. Giard DJ, Fleischaker RJ, Fabrikant M. Effect of temperature on the production of human fibroblast interferon (41411) Proc Soc Exp Biol Med. 1982;170:155–159. doi: 10.3181/00379727-170-41411. [DOI] [PubMed] [Google Scholar]
  8. Goergen JL, Marc A, Engasser JM. Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release. Cytotechnology. 1993;11:189–195. doi: 10.1007/BF00749869. [DOI] [PubMed] [Google Scholar]
  9. Gorczyca W, Bigman K, Mittleman A, Ahmed T, Gong J, Melamed MR, Darzynkiewicz Z. Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia. 1993;7:659–670. [PubMed] [Google Scholar]
  10. Gorczyca W, Bruno S, Darzynkiewicz RJ, Gong J, Darzynkiewicz Z. 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. 1992;1:639–648. doi: 10.3892/ijo.1.6.639. [DOI] [PubMed] [Google Scholar]
  11. Jenkins N, Hovey A. Temperature control of growth and productivity in mutant Chinese hamster ovary cells synthesizing a recombinant protein. Biotechnology and Bioengineering. 1993;42:1029–1036. doi: 10.1002/bit.260420903. [DOI] [PubMed] [Google Scholar]
  12. Kaufman RJ, Sharp PA. Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene. J Mol Biol. 1982;159:601–621. doi: 10.1016/0022-2836(82)90103-6. [DOI] [PubMed] [Google Scholar]
  13. Moore A, Donahue CJ, Hooley J, Stocks DL, Bauer KD, Mather JP. Apoptosis in CHO cell batch cultures: examination by flow cytometry. Cytotechnology. 1995;17:1–11. doi: 10.1007/BF00749215. [DOI] [PubMed] [Google Scholar]
  14. Müller M, Siems W, Buttgereit F, Dumdey, Rapoport SM. Quantification of ATP-producing and consuming processes of Ehrlich ascites tumor cells. Eur J Biochem. 1986;161:701–705. doi: 10.1111/j.1432-1033.1986.tb10496.x. [DOI] [PubMed] [Google Scholar]
  15. Ramírez OT, Mutharasan R. Cell cycle-and growth phase — dependent variations in size distribution, antibody productivity and oxygen demand in hybridoma cultures. Biotechnol Bioeng. 1990;36:839–848. doi: 10.1002/bit.260360814. [DOI] [PubMed] [Google Scholar]
  16. Reuveny S, Velez D, Maxmillan JD, Miller L. Factors affecting cell growth and monoclonal antibody production in stirred reactors. J Immunol Methods. 1986;86:53–59. doi: 10.1016/0022-1759(86)90264-4. [DOI] [PubMed] [Google Scholar]
  17. Reuveny S, Kim YJ, Kemp CW, Shiloach J. Effect of temperature and oxygen on cell growth and recombinant protein production in insect cell cultures. Appl. Microbiol Biotechnol. 1993;38:619–623. doi: 10.1007/BF00182800. [DOI] [PubMed] [Google Scholar]
  18. Ryll T, Wagner R. 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. 1991;570:77–88. doi: 10.1016/0378-4347(91)80202-n. [DOI] [PubMed] [Google Scholar]
  19. Ryll T, Wagner R. Intracellular ribonucleotide pools as a tool for monitoring the physiological state of in vitro cultivated mammalian cells during production processes. Biotechnology and Bioengineering. 1992;40:934–946. doi: 10.1002/bit.260400810. [DOI] [PubMed] [Google Scholar]
  20. Ryll T, Valley U, Wagner R. Biochemistry of growth inhibition by ammonium ions in mammalian cells. Biotechnology and Bioengineering. 1994;44:184–193. doi: 10.1002/bit.260440207. [DOI] [PubMed] [Google Scholar]
  21. Schwartzman RA, Cidlowski JA. Mechanism of tissue-specific induction of internucleosomal deoxyribonucleic acid cleavage activity and apoptosis by glucocorticoids. Endocrinology. 1993;133:591–599. doi: 10.1210/endo.133.2.8393769. [DOI] [PubMed] [Google Scholar]
  22. Singh RP, Al-Rubeai M, Gregory CD, Emery AN. Cell death in bioreactors: a role for apoptosis. Biotechnol Bioeng. 1994;44:720–726. doi: 10.1002/bit.260440608. [DOI] [PubMed] [Google Scholar]
  23. Singh M, Al-Rubeai M, Goldman MH, Emery AN. Apoptosis — the programme for cell death in agitated bioreactors. Biotechnol. 1994;94(ABE2):23–25. [Google Scholar]
  24. Sureshkumar GK, Muthaeasan R. The influence of temperature on a mouse-mouse hybridoma growth and monoclonal antibody production. Biotechnol Bioeng. 1991;37:292–295. doi: 10.1002/bit.260370313. [DOI] [PubMed] [Google Scholar]
  25. Suzuki E, Ollis DF. Cell cycle model for antibody production kinetics. Biotechn Bioeng. 1989;34:1398. doi: 10.1002/bit.260341109. [DOI] [PubMed] [Google Scholar]
  26. Telford WG, King LE, Fraker PJ. Evaluation of glucocorticoid-induced DNA fragmentation in mouse thymocytes by flow cytometry. Cell Prolif. 1991;24:447–459. doi: 10.1111/j.1365-2184.1991.tb01173.x. [DOI] [PubMed] [Google Scholar]
  27. Tilly JL, Hsueh AJW. Microscale autoradiographic method for the quanlitative and quantitative analysis of apoptotic DNA fragmentation. J Cell Physiol. 1993;154:519–626. doi: 10.1002/jcp.1041540310. [DOI] [PubMed] [Google Scholar]
  28. Urlaub G, Chasin LA. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci USA. 1980;77:4216–4220. doi: 10.1073/pnas.77.7.4216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Weidemann R, Ludwig A, Kretzmer G. Low temperature cultivation — A step towards process optimization. Cytotechnology. 1994;15:111–116. doi: 10.1007/BF00762385. [DOI] [PubMed] [Google Scholar]
  30. Zaharevitz DA, Napier EA, Anderson LW, Strong JM, Cysyk RL. Stimulation of uracil nucleotide synthesis in mouse liver, intestine and kidney by ammonium chloride infusion. European Journal of Biochemistry. 1988;175:193–198. doi: 10.1111/j.1432-1033.1988.tb14183.x. [DOI] [PubMed] [Google Scholar]

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