Cytotechnology

, Volume 64, Issue 4, pp 429–441 | Cite as

Real-time monitoring of adherent Vero cell density and apoptosis in bioreactor processes

  • Emma Petiot
  • Amal El-Wajgali
  • Geoffrey Esteban
  • Cécile Gény
  • Hervé Pinton
  • Annie Marc
Original Research

Abstract

This study proposes an easy to use in situ device, based on multi-frequency permittivity measurements, to monitor the growth and death of attached Vero cells cultivated on microporous microcarriers, without any cell sampling. Vero cell densities were on-line quantified up to 106 cell mL−1. Some parameters which could potentially impact Vero cell morphological and physiological states were assessed through different culture operating conditions, such as media formulation or medium feed-harvest during cell growth phase. A new method of in situ cell death detection with dielectric spectroscopy was also successfully implemented. Thus, through permittivity frequency scanning, major rises of the apoptotic cell population in bioreactor cultures were detected by monitoring the characteristic frequency of the cell population, fc, which is one of the culture dielectric parameters. Both cell density quantification and cell apoptosis detection are strategic information in cell-based production processes as they are involved in major events of the process, such as scale-up or choice of the viral infection conditions. This new application of dielectric spectroscopy to adherent cell culture processes makes it a very promising tool for risk-mitigation strategy in industrial processes. Therefore, our results contribute to the development of Process Analytical Technology in cell-based industrial processes.

Keywords

Adherent Vero cells In situ monitoring Multi-frequency permittivity Cell density Apoptosis 

References

  1. Al-Rubeai M (1998) Apoptosis and cell culture technology. Adv Biochem Eng Biotechnol 59:225–249CrossRefGoogle Scholar
  2. Ansorge S, Esteban G, Schmid G (2007) On-line monitoring of infected Sf-9 insect cell cultures by scanning permittivity measurements and comparison with off-line biovolume measurements. Cytotechnology 55:115–124CrossRefGoogle Scholar
  3. Ansorge S, Esteban G, Schmid G (2010) Multifrequency permittivity measurements enable on-line monitoring of changes in intracellular conductivity due to nutrient limitations during batch cultivations of CHO cells. Biotechnol Prog 26:272–283Google Scholar
  4. Ansorge S, Lanthier S, Transfiguracion J, Henry O, Kamen A (2011) Monitoring lentiviral vector production kinetics using online permittivity measurements. Biochem Eng J 54:16–25Google Scholar
  5. Bradamante S, Barenghi L, Villa A (2004) Simulated weightlessness in the design and exploitation of a NMR-compatible bioreactor. Biotechnol Prog 20:1454–1459CrossRefGoogle Scholar
  6. Butler M, Burgener A, Patrick M, Berry M, Moffatt D, Huzel N, Barnabe N, Coombs K (2000) Application of a serum-free medium for the growth of vero cells and the production of reovirus. Biotechnol Prog 16:854–858CrossRefGoogle Scholar
  7. Cannizzaro C, Gügerli R, Marison I, Stockar Uv (2003) On-line biomass monitoring of CHO perfusion culture with scanning dielectric spectroscopy. Biotechnol Bioeng 84:597–610CrossRefGoogle Scholar
  8. Card C, Hunsaker B, Smith T, Hirsch J (2008). Near-infrared spectroscopy for rapid, simultaneous monitoring of multiple components in mammalian cell culture. BioProc Int 6:58–67Google Scholar
  9. Chan YF, Abubakar S (2003) Enterovirus 71 infection induces apoptosis in Vero cells. Malays J Pathol 25:29–35Google Scholar
  10. Conlon I, Lloyd A, Raff M (2004) Coordination of cell growth and cell cycle progression in proliferating mammalian cells. In: Hall MN, Raff M, Thomas G (eds) Cell growth: control of cell size. Laboratory Press, Cold Spring Harbor. pp 85–101Google Scholar
  11. Ducommun P, Bolzonella I, Rhiel M, Pugeaud P, von Stockar U, Marison IW (2001) On-line determination of animal cell concentration. Biotechnol Bioeng 72:515–522CrossRefGoogle Scholar
  12. Ducommun P, Kadouri A, von Stockar U, Marison IW (2002) On-line determination of animal cell concentration in two industrial high-density culture processes by dielectric spectroscopy. Biotechnol Bioeng 77:316–323CrossRefGoogle Scholar
  13. Eyer K, Heinzle E (1996) On-line estimation of viable cells in a hybridoma culture at various DO levels using ATP balancing and redox potential measurement. Biotechnol Bioeng 49:277–283CrossRefGoogle Scholar
  14. Figueroa B, Chen S, Oyler GA, Hardwick JM, Betenbaugh MJ (2004) Aven and Bcl-xL enhance protection against apoptosis for mammalian cells exposed to various culture conditions. Biotechnol Bioeng 85:589–600CrossRefGoogle Scholar
  15. Gentet LJ, Stuart GJ, Clements JD (2000) Direct measurement of specific membrane capacitance in neurons. Biophys J 79:314–320CrossRefGoogle Scholar
  16. Huang C, Chen A, Wang L, Guo M, Yu J (2007) Electrokinetic measurements of dielectric properties of membrane for apoptotic HL-60 cells on chip-based device. Biomed Microdevices 9:335–343CrossRefGoogle Scholar
  17. Ishaque A, Al-Rubeai M (1998) Use of intracellular pH and annexin-V flow cytometric assays to monitor apoptosis and its suppression by bcl-2 over-expression in hybridoma cell culture. J Immunol Methods 221:43–57CrossRefGoogle Scholar
  18. Kamen AA, Bédard C, Tom R, Perret S, Jardin B (1996) On-line monitoring of respiration in recombinant-baculovirus infected and uninfected insect cell bioreactor cultures. Biotechnol Bioeng 50:36–48CrossRefGoogle Scholar
  19. Kanapitsas A, Vartzeli-Nikaki P, Konsta AA, Visvardis EE, Sideris EG (2006) Dielectric properties during apoptosis in peripheral blood cells from chronic lymphocytic leukaemia patients. Dielectr Electr Insulation IEEE Trans 13:1057–1062Google Scholar
  20. Kilburn DG, Fitzpatrick P, Blake-Coleman BC, Clarke DJ, Griffiths JB (1989) On-line monitoring of cell mass in mammalian cell cultures by acoustic densitometry. Biotechnol Bioeng 33:1379–1384CrossRefGoogle Scholar
  21. Kistner O, Barrett PN, Mundt W, Reiter M, Schober-Bendixen S, Dorner F (1998) Development of a mammalian cell (Vero) derived candidate influenza virus vaccine. Vaccine 16:960–968CrossRefGoogle Scholar
  22. Knezevic I, Stacey G, Petricciani J (2008) WHO Study Group on cell substrates for production of biologicals, Geneva, Switzerland, 11–12 June 2007. Biologicals 36:203–211CrossRefGoogle Scholar
  23. Konstantinov K, Chuppa S, Sajan E, Tsai Y, Yoon S, Golini F (1994) Real-time biomass-concentration monitoring in animal-cell cultures. Trends Biotechnol 12:324–333CrossRefGoogle Scholar
  24. Labeed FH, Coley HM, Hughes MP (2006) Differences in the biophysical properties of membrane and cytoplasm of apoptotic cells revealed using dielectrophoresis. Biochim Biophys Acta 1760: 922–929Google Scholar
  25. Le Ru A, Jacob D, Transfiguracion J, Ansorge S, Henry O, Kamen A (2010) Scalable production of influenza virus in HEK-293 cells for efficient vaccine manufacturing. Vaccine 28:3661–3671CrossRefGoogle Scholar
  26. Liu C–C, Lian W-C, Butler M, Wu S-C (2007) High immunogenic enterovirus 71 strain and its production using serum-free microcarrier Vero cell culture. Vaccine 25:19–24CrossRefGoogle Scholar
  27. Mandenius C-F, Graumann K, Schultz TW, Premstaller A, Olsson I-M, Petiot E, Clemens C, Welin M (2009) Quality by design (QbD) for biotechnology-related pharmaceuticals. Biotechnol J 4:600–609CrossRefGoogle Scholar
  28. Markx GH, Davey CL (1999) The dielectric properties of biological cells at radiofrequencies: applications in biotechnology. Enzym Microb Technol 25:161–171CrossRefGoogle Scholar
  29. Matanguihan RM, Konstantinov KB, Yoshida T (1994) Dielectric measurement to monitor the growth and the physiological states of biological cells. Bioproc Biosyst Eng 11:213–222Google Scholar
  30. Merten OW (2000) Safety for vaccine(e)s. Cytotechnology 34:181–183CrossRefGoogle Scholar
  31. Merten O-W (2009) Cell detachment. In: Flickinger MC (ed) Encyclopedia of industrial biotechnology : bioprocess, bioseparation, and cell technology. Wiley, New York. pp 1–22Google Scholar
  32. Noll T, Biselli M (1998) Dielectric spectroscopy in the cultivation of suspended and immobilized hybridoma cells. J Biotechnol 63:187–198CrossRefGoogle 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 26:1187–1199Google Scholar
  34. Patel PM, Markx GH (2008) Dielectric measurement of cell death. Enzym Microb Technol 43:463–470CrossRefGoogle Scholar
  35. Patel PM, Bhat A, Markx GH (2008) A comparative study of cell death using electrical capacitance measurements and dielectrophoresis. Enzym Microb Technol 43:523–530CrossRefGoogle Scholar
  36. Pethig R, Jakubek L, Sanger R, Heart E, Corson E, Smith P (2005) Electrokinetic measurements of membrane capacitance and conductance for pancreatic beta-cells. IEE Proc Nanobiotechnol 152:189–193CrossRefGoogle Scholar
  37. Petiot E, Fournier F, Gény C, Pinton H, Marc A (2010a) Rapid screening of serum-free media for the growth of adherent vero cells by using a small-scale and non-invasive tool. Appl Biochem Biotechnol 160:1600–1615CrossRefGoogle Scholar
  38. Petiot E, Guedon E, Blanchard F, Gény C, Pinton H, Marc A (2010b) Kinetic characterization of vero cell metabolism in a serum-free batch culture process. Biotechnol Bioeng 107:143–153CrossRefGoogle Scholar
  39. Petiot E, Jacob D, Lanthier S, Lohr V, Ansorge S, Kamen A (2011) Metabolic and kinetic analyses of influenza production in perfusion HEK293 cell culture. BMC Biotechnol 11:84CrossRefGoogle Scholar
  40. Pons M-N, Wagner A, Vivier H, Marc A (1992) Application of quantitative image analysis to a mammalian cell line grown on microcarriers. Biotechnol Bioeng 40:187–193CrossRefGoogle Scholar
  41. Ravindra PV, Tiwari AK, Ratta B, Chaturvedi U, Palia SK, Subudhi PK, Kumar R, Sharma B, Rai A, Chauhan RS (2008) Induction of apoptosis in Vero cells by Newcastle disease virus requires viral replication, de novo protein synthesis and caspase activation. Virus Res 133:285–290CrossRefGoogle Scholar
  42. Rourou S, van der Ark A, van der Velden T, Kallel HA (2007) A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditions. Vaccine 25:3879–3889CrossRefGoogle Scholar
  43. Rudolph G, Lindner P, Gierse A, Bluma A, Martinez G, Hitzmann B, Scheper T (2008) Online monitoring of microcarrier based fibroblast cultivations with in situ microscopy. Biotechnol Bioeng 99:136–145CrossRefGoogle Scholar
  44. Santos-Sacchi J, Navarrete E (2002) Voltage-dependent changes in specific membrane capacitance caused by prestin, the outer hair cell lateral membrane motor. Pflügers Archiv Eur J Physiol 444:99–106CrossRefGoogle Scholar
  45. Schulze-Horsel J, Schulze M, Agalaridis G, Genzel Y, Reichl U (2009) Infection dynamics and virus-induced apoptosis in cell culture-based influenza vaccine production–flow cytometry and mathematical modeling. Vaccine 27:2712–2722CrossRefGoogle Scholar
  46. Schwan HP (1957) Electrical properties of tissue and cell suspensions. Adv Biol Med Phys 5:147–208Google Scholar
  47. Shah D, Clee P, Boisen S, Al-Rubai M (2006) NucleoCounter–an efficient technique for the determination of cell number and viability in animal cell culture processes. Cytotechnology 51:39–44Google Scholar
  48. Siano SA (1997) Biomass measurement by inductive permittivity. Biotechnol Bioeng 55:289–304CrossRefGoogle Scholar
  49. Souza M, Freire M, Castilho L (2007) Cultivation of vero cells on microporous and macroporous microcarriers. In: Smith R (ed) Cell technology for cell products. Springer, Harrogate, UK, pp 753–755Google Scholar
  50. Souza MCO, Freire MS, Schulze EA, Gaspar LP, Castilho LR (2009) Production of yellow fever virus in microcarrier-based Vero cell cultures. Vaccine 27:6420–6423CrossRefGoogle Scholar
  51. Szabo S, Monroe S, Fiorino S, Bitzan J, Loper K (2004) Evaluation of an automated instrument for viability and concentration measurements of cryopreserved hematopoetic cells. Lab Hematol 10:109–111CrossRefGoogle Scholar
  52. Teixeira AP, Portugal CAM, Carinhas N, Dias JML, Crespo JP, Alves PM, Carrondo MJT, Oliveira R (2009) In situ 2D fluorometry and chemometric monitoring of mammalian cell cultures. Biotechnol Bioeng 102:1098–1106CrossRefGoogle Scholar
  53. Toriniwa H, Komiya T (2007) Japanese encephalitis virus production in Vero cells with serum-free medium using a novel oscillating bioreactor. Biologicals 35:221–226CrossRefGoogle Scholar
  54. Toriniwa H, Komiya T (2008) Long-term stability of Vero cell-derived inactivated Japanese encephalitis vaccine prepared using serum-free medium. Vaccine 26:3680–3689CrossRefGoogle Scholar
  55. Umegaki R, Kino-oka M, Taya M (2004) Assessment of cell detachment and growth potential of human keratinocyte based on observed changes in individual cell area during trypsinization. Biochem Eng J 17:49–55CrossRefGoogle Scholar
  56. Wang X, Becker FF, Gascoyne PRC (2002) Membrane dielectric changes indicate induced apoptosis in HL-60 cells more sensitively than surface phosphatidylserine expression or DNA fragmentation. Biochem Biophys Acta 1564:412–420 Google Scholar
  57. Zeiser A, Bédard C, Voyer R, Jardin B, Tom R, Kamen AA (1999) On-line monitoring of the progress of infection in Sf-9 insect cell cultures using relative permittivity measurements. Biotechnol Bioeng 63:122–126CrossRefGoogle Scholar
  58. Zeiser A, Elias CB, Voyer R, Jardin B, Kamen AA (2000) On-line monitoring of physiological parameters of insect cell cultures during the growth and infection process. Biotechnol Prog 16:803–808CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Emma Petiot
    • 1
    • 4
  • Amal El-Wajgali
    • 1
  • Geoffrey Esteban
    • 2
  • Cécile Gény
    • 3
  • Hervé Pinton
    • 3
  • Annie Marc
    • 1
  1. 1.Laboratoire Réactions et Génie des Procédés, UPR CNRS 3349Nancy-UniversitéVandoeuvre-lès-Nancy CedexFrance
  2. 2.FOGALE NanotechNîmesFrance
  3. 3.Sanofi PasteurMarcy L’EtoileFrance
  4. 4.Animal Cell Technology Group, Biotechnology Research InstituteMontrealCanada

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