Skip to main content
SpringerLink
Log in

Impact of apoptosis on the on-line measured dielectric properties of CHO cells

  • Original Paper
  • Published:
Bioprocess and Biosystems Engineering Aims and scope Submit manuscript

Abstract

Apoptosis is a common type of cell death in biopharmaceutical cell culture processes which causes decrease in viable cell density and product yield. The progression of apoptosis has been reported to influence the dielectric properties of mammalian cells; however, the on-line detection of these effects has been rarely described. This study provides a comprehensive analysis of the on-line detectability of dielectric changes upon apoptosis induction in an industrial fed-batch process of CHO cells expressing a recombinant monoclonal antibody. Using capacitance signals, measured at 25 frequencies, the impact of apoptosis on the dielectric spectra was investigated in eight bioreactor cultivations in which various process conditions were combined with two different apoptosis induction strategies (camptothecin treatment and glucose starvation). To differentiate the apoptosis-related information from the cell concentration-associated variance in the multivariate capacitance datasets, principal component analysis (PCA) was used. A second principal component, explaining an explicit proportion (>20 %) of the variance, was identified to be related to dielectric changes induced by apoptosis. Furthermore, the analysis of caspase-3 and -7 activation and DNA fragmentation showed that the detected dielectric change occurred in the early phase of apoptosis. The presented results verify that apoptosis has a considerable impact on the dielectric features of CHO cells and it can be monitored on-line with the introduced tool-set combining capacitance measurement with multivariate data analysis.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Edinger AL, Thompson CB (2004) Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol 16:663–669. doi:10.1016/j.ceb.2004.09.011

    Article  CAS  Google Scholar 

  2. Kanduc D, Mittelman A, Serpico R et al (2002) Cell death: apoptosis versus necrosis (review). Int J Oncol 21:165–170

    CAS  Google Scholar 

  3. Kurokawa M, Kornbluth S (2009) Caspases and kinases in a death grip. Cell 138:838–854. doi:10.1016/j.cell.2009.08.021

    Article  CAS  Google Scholar 

  4. Häcker G (2000) The morphology of apoptosis. Cell Tissue Res 301:5–17. doi:10.1007/s004410000193

    Article  Google Scholar 

  5. Cotter TG, al-Rubeai M (1995) Cell death (apoptosis) in cell culture systems. Trends Biotechnol 13:150–155. doi:10.1016/S0167-7799(00)88926-X

    Article  CAS  Google Scholar 

  6. Nicotera P, Leist M, Ferrando-May E (1998) Intracellular ATP, a switch in the decision between apoptosis and necrosis. Toxicol Lett pp 139–142

  7. Bortner CD, Oldenburg BEN, Cidlowski JA, Oldenburg NBE (1995) The role of DNA fragmentation in apoptosis. Trends Cell Biol 5:21–26. doi:10.1016/S0962-8924(00)88932-1

    Article  CAS  Google Scholar 

  8. Enari M, Sakahira H, Yokoyama H et al (1998) A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391:43–50

    Article  CAS  Google Scholar 

  9. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35:495–516

    Article  CAS  Google Scholar 

  10. Arden N, Betenbaugh MJ (2004) Life and death in mammalian cell culture: strategies for apoptosis inhibition. Trends Biotechnol 22:174–180

    Article  CAS  Google Scholar 

  11. Goswami J, Sinskey AJ, Steller H et al (1999) Apoptosis in batch cultures of Chinese hamster ovary cells. Biotechnol Bioeng 62:632–640

    Article  CAS  Google Scholar 

  12. Singh RP, Finka G, Emery AN (1997) Apoptosis and its control in cell culture systems. 87–93

  13. Arden N, Betenbaugh MJ (2006) Regulating apoptosis in mammalian cell cultures. Cytotechnology 50:77–92. doi:10.1007/s10616-006-9008-5

    Article  CAS  Google Scholar 

  14. Krampe B, Al-Rubeai M (2010) Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies. Cytotechnology 62:175–188. doi:10.1007/s10616-010-9274-0

    Article  Google Scholar 

  15. Mercille S, Massie B (1994) Induction of apoptosis in oxygen-deprived cultures of hybridoma cells. Cytotechnology 15:117–128

    Article  CAS  Google Scholar 

  16. Majid FAA, Butler M, Al-Rubeai M (2007) Glycosylation of an immunoglobulin produced from a murine hybridoma cell line: the effect of culture mode and the anti-apoptotic gene, bcl-2. Biotechnol Bioeng 97:156–169. doi:10.1002/bit.21207

    Article  CAS  Google Scholar 

  17. Kapuy O, Vinod PK, Mandl J, Bánhegyi G (2013) A cellular stress-directed bistable switch controls the crosstalk between autophagy and apoptosis. Mol BioSyst 9:296–306. doi:10.1039/c2mb25261a

    Article  CAS  Google Scholar 

  18. Geske FJ, Lieberman R, Strange R, Gerschenson LE (2001) Early stages of p53-induced apoptosis are reversible. Cell Death Differ 8:182–191. doi:10.1038/sj.cdd.4400786

    Article  CAS  Google Scholar 

  19. Druzinec D, Weiss K, Elseberg C et al (2014) Animal Cell Biotechnology. In: Pörtner R (ed) Humana Press, Totowa, pp 313–341

  20. Asami K (2002) Characterization of biological cells by dielectric spectroscopy. J Non Cryst Solids 305:268–277. doi:10.1016/S0022-3093(02)01110-9

    Article  CAS  Google Scholar 

  21. Davey CL, Davey HM, Kell DB, Todd RW (1993) Introduction to the dielectric estimation of cellular biomass in real time, with special emphasis on measurements at high volume fractions. Anal Chim Acta 279:155–161. doi:10.1016/0003-2670(93)85078-X

    Article  Google Scholar 

  22. Carvell JP, Dowd JE (2006) On-line measurements and control of viable cell density in cell culture manufacturing processes using radio-frequency impedance. Cytotechnology 50:35–48. doi:10.1007/s10616-005-3974-x

    Article  CAS  Google Scholar 

  23. Chin S, Hughes MP, Coley HM, Labeed FH (2006) Rapid assessment of early biophysical changes in K562 cells during apoptosis determined using dielectrophoresis. IntJNanomedicine 1:333–337

    CAS  Google Scholar 

  24. Braasch K, Nikolic-Jaric M, Cabel T et al (2013) The changing dielectric properties of CHO cells can be used to determine early apoptotic events in a bioprocess. BiotechnolBioeng 110:2902–2914

    CAS  Google Scholar 

  25. Herrmann M, Lorenz HM, Voll R et al (1994) A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Res 22:5506–5507

    Article  CAS  Google Scholar 

  26. Dabros M, Dennewald D, Currie DJ et al (2009) Cole–Cole, linear and multivariate modeling of capacitance data for on-line monitoring of biomass. Bioprocess Biosyst Eng 32:161–173. doi:10.1007/s00449-008-0234-4

    Article  CAS  Google Scholar 

  27. Pommier Y (2006) Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer 6:789–802. doi:10.1038/nrc1977

    Article  CAS  Google Scholar 

  28. King MA, Radicchi-Mastroianni MA (2002) Effects of caspase inhibition on camptothecin-induced apoptosis of HL-60 cells. Cytometry 49:28–35. doi:10.1002/cyto.10141

    Article  CAS  Google Scholar 

  29. Hsiang YH, Hertzberg R, Hecht S, Liu LF (1985) Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 260:14873–14878

    CAS  Google Scholar 

  30. Hinz JM, Helleday T, Meuth M (2003) Reduced apoptotic response to camptothecin in CHO cells deficient in XRCC3 DNA repair, cell-cycle arrest and apoptosis Several reports suggest that such responses may be coordinated by com-signalling other cellular responses. The Rad51-guided homolog. 24:249–253

  31. Cannizzaro C, Gügerli R, Marison I, Von Stockar U (2003) On-line biomass monitoring of CHO perfusion culture with scanning dielectric spectroscopy. Biotechnol Bioeng 84:597–610. doi:10.1002/bit.10809

    Article  CAS  Google Scholar 

  32. Ansorge S, Esteban G, Schmid G (2010) On-line monitoring of responses to nutrient feed additions by multi-frequency permittivity measurements in fed-batch cultivations of CHO cells. Cytotechnology 62:121–132. doi:10.1007/s10616-010-9267-z

    Article  CAS  Google Scholar 

  33. Opel CF, Li J, Amanullah A (2010) Quantitative modeling of viable cell density, cell size, intracellular conductivity, and membrane capacitance in batch and fed-batch CHO processes using dielectric spectroscopy. Biotechnol Prog 26:1187–1199. doi:10.1002/btpr.425

    CAS  Google Scholar 

  34. Downey BJ, Graham LJ, Breit JF, Glutting NK (2014) A novel approach for using dielectric spectroscopy to predict viable cell volume (VCV) in early process development. Biotechnol Prog 30:479–487. doi:10.1002/btpr.1845

    Article  CAS  Google Scholar 

  35. Petiot E, El-Wajgali A, Esteban G et al (2012) Real-time monitoring of adherent Vero cell density and apoptosis in bioreactor processes. Cytotechnology 64:429–441. doi:10.1007/s10616-011-9421-2

    Article  CAS  Google Scholar 

  36. Schwamb S, Munteanu B, Meyer B et al (2013) Monitoring CHO cell cultures: cell stress and early apoptosis assessment by mass spectrometry. J Biotechnol 168:452–461. doi:10.1016/j.jbiotec.2013.10.014

    Article  CAS  Google Scholar 

  37. Mulukutla BC, Khan S, Lange A, Hu W-S (2010) Glucose metabolism in mammalian cell culture: new insights for tweaking vintage pathways. Trends Biotechnol 28:476–484. doi:10.1016/j.tibtech.2010.06.005

    Article  CAS  Google Scholar 

  38. Majors BS, Betenbaugh MJ, Chiang GG (2007) Links between metabolism and apoptosis in mammalian cells: applications for anti-apoptosis engineering. Metab Eng 9:317–326. doi:10.1016/j.ymben.2007.05.003

    Article  CAS  Google Scholar 

  39. Sun OH, Lee GM (2008) Nutrient deprivation induces autophagy as well as apoptosis in Chinese hamster ovary cell culture. Biotechnol Bioeng 99:678–685. doi:10.1002/bit.21589

    Article  Google Scholar 

Download references

Acknowledgments

We gratefully acknowledge Christoph Herwig (Vienna University of Technology, Austria), Erik Bogsch, and László Párta (Gedeon Richter Plc., Budapest, Hungary) for their critical review of this manuscript. We thank Dóra Molnár for the operational assistance in cell cultivation and the bioanalytical group at Gedeon Richter Plc. for their assistance in the analytical measurements.

Author information

Authors and Affiliations

  1. Department of Biotechnology, Gedeon Richter Plc, 19-21, Gyömrői út, Budapest, 1103, Hungary

    Dénes Zalai, Teodóra Tobak & Ákos Putics

Authors
  1. Dénes Zalai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Teodóra Tobak
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ákos Putics
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ákos Putics.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 150 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zalai, D., Tobak, T. & Putics, Á. Impact of apoptosis on the on-line measured dielectric properties of CHO cells. Bioprocess Biosyst Eng 38, 2427–2437 (2015). https://doi.org/10.1007/s00449-015-1479-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-015-1479-3

Keywords

Search

Navigation