Applied Microbiology and Biotechnology

, Volume 73, Issue 6, pp 1423–1434 | Cite as

Hybridoma Ped-2E9 cells cultured under modified conditions can sensitively detect Listeria monocytogenes and Bacillus cereus

  • Pratik Banerjee
  • Mark T. Morgan
  • Jenna L. Rickus
  • Kathy Ragheb
  • Carlos Corvalan
  • J. Paul Robinson
  • Arun K. Bhunia
Applied Microbial and Cell Physiology


Lymphocyte origin hybridoma Ped-2E9 cell-based cytotoxicity assay can detect virulent Listeria or Bacillus species, and its application in a cell-based biosensor for onsite use would be very attractive. However, maintaining enough viable cells on a sensor platform for a prolonged duration is a challenging task. In this study, key factors affecting the survival and growth of Ped-2E9 cells under modified conditions were investigated. When the Ped-2E9 cells were grown in media containing 5% fetal bovine serum in sealed tubes without any replenishment of nutrients or exogenous CO2 supply, a large portion of the cells remained viable for 6 to 7 days and cells entered into G0/G1 resting phase. The media pH change was negligible and no cell death was observed in the first 4 days, then cells sequentially underwent apoptotic (fourth day onward) phase until day 7 after which a majority was dead. Subsequent cytotoxicity testing of 3- to 7-day stored Ped-2E9 cells sensitively detected virulent Listeria and Bacillus species. These data strongly suggest that Ped-2E9 cells can be maintained in viable state for 6 days in a sealed tube mimicking the environment in a potential sensor device for onsite use without the need for expensive cell culture facilities.


Ped-2E9 Hybridoma Listeria monocytogenes Bacillus Cytotoxicity Cell-based sensor 



A part of this research was supported through a cooperative agreement with the Agricultural Research Service of the US Department of Agriculture (USDA) project number 1935-42000-035, the center for Food Safety and Engineering at Purdue University, and USDA-NRI (2005-35603-16338) awarded to JLR and AKB.


  1. al-Rubeai M, Emery AN, Chalder S (1992a) The effect of Pluronic F-68 on hybridoma cells in continuous culture. Appl Microbiol Biotechnol 37:44–55CrossRefGoogle Scholar
  2. al-Rubeai M, Emery AN, Chalder S, Jan DC (1992b) Specific monoclonal antibody productivity and the cell cycle-comparisons of batch, continuous and perfusion cultures. Cytotechnology 9:85–97CrossRefGoogle Scholar
  3. Baker P, Knoblock K, Noll L, Wyatt D, Lydersen B (1985) A serum independent medium effective in all aspects of hybridoma technology and immunological applications. Dev Biol Stand 60:63–72Google Scholar
  4. Bhunia AK, Feng X (1999) Examination of cytopathic effect and apoptosis in Listeria monocytogenes-infected hybridoma B-lymphocyte (Ped-2E9) line in vitro. J Microbiol Biotechnol 9:398–403Google Scholar
  5. Bhunia AK, Westbrook DG (1998) Alkaline phosphatase release assay to determine cytotoxicity for Listeria species. Lett Appl Microbiol 26:305–310CrossRefGoogle Scholar
  6. Bhunia AK, Steele PJ, Westbrook DG, Bly LA, Maloney TP, Johnson MG (1994) A six-hour in vitro virulence assay for Listeria monocytogenes using myeloma and hybridoma cells from murine and human sources. Microb Pathog 16:99–110CrossRefGoogle Scholar
  7. Bhunia AK, Westbrook DG, Story R, Johnson MG (1995) Frozen stored murine hybridoma cells can be used to determine the virulence of Listeria monocytogenes. J Clin Microbiol 33:3349–3351Google Scholar
  8. Borth N, Heider R, Assadian A, and Katinger H (1992) Growth and production kinetics of human x mouse and mouse hybridoma cells at reduced temperature and serum content. J Biotechnol 25:319–331CrossRefGoogle Scholar
  9. Butler M, Huzel N (1995) The effect of fatty acids on hybridoma cell growth and antibody productivity in serum-free cultures. J Biotechnol 39:165–173CrossRefGoogle Scholar
  10. Compton RS, Konigsberg IR (1988) Cell cycle withdrawal without concomitant differentiation: analysis of a G1-specific temperature-sensitive murine myoblast cell line. Dev Biol 129:476–494CrossRefGoogle Scholar
  11. Cooper S (2003) Reappraisal of serum starvation, the restriction point, G0, and G1 phase arrest points. FASEB J 17:333–340CrossRefGoogle Scholar
  12. Dedov VN, Dedova IV, Nicholson GA (2004) Equilibrium between cell division and apoptosis in immortal cells as an alternative to the G1 restriction mechanism in mammalian cells. Cell Cycle 3:491–495Google Scholar
  13. Deng J, Schoenbach KH, Buescher ES, Hair PS, Fox PM, Beebe SJ (2003) The effects of intense submicrosecond electrical pulses on cells. Biophys J 84:2709–2714CrossRefGoogle Scholar
  14. deZengotita VM, Abston LR, Schmelzer AE, Shaw S, Miller WM (2002) Selected amino acids protect hybridoma and CHO cells from elevated carbon dioxide and osmolality. Biotechnol Bioeng 78:741–752CrossRefGoogle Scholar
  15. Farber JM, Speirs JI (1987) Potential use of continuous cell lines to distinguish between pathogenic and nonpathogenic Listeria spp. J Clin Microbiol 25:1463–1466Google Scholar
  16. Fassnacht D, Rossing S, Ghaussy N, Portner R (1997) Influence of non-essential amino acids on apoptotic and necrotic death of mouse hybridoma cells in batch cultures. Biotechnol Lett 19:35–38CrossRefGoogle Scholar
  17. Franek F, Dolnikova J (1991) Hybridoma growth and monoclonal antibody production in iron-rich protein-free medium: effect of nutrient concentration. Cytotechnology 7:33–38CrossRefGoogle Scholar
  18. Franek F, Sramkova K (1996) Protection of B lymphocyte hybridoma against starvation-induced apoptosis: survival–signal role of some amino acids. Immunol Lett 52:139–144CrossRefGoogle Scholar
  19. Froud SJ (1999) The development, benefits and disadvantages of serum-free media. Dev Biol Stand 99:157–166Google Scholar
  20. Fukuda N, Saitoh M, Kobayashi N, Miyazono K (2006) Execution of BMP-4-induced apoptosis by p53-dependent ER dysfunction in myeloma and B-cell hybridoma cells. Oncogene 25(25):3509–3517CrossRefGoogle Scholar
  21. Giaever I, Keese CR (1993) A morphological biosensor for mammalian cells. Nature 366:591–592CrossRefGoogle Scholar
  22. Gracieux P, Roche SM, Pardon P, Velge P (2003) Hypovirulent Listeria monocytogenes strains are less frequently recovered than virulent strains on PALCAM and Rapid′ L. mono media. Int J Food Microbiol 83:133–145CrossRefGoogle Scholar
  23. Gray KM, Bhunia AK (2005) Specific detection of cytopathogenic Listeria monocytogenes using a two-step method of immunoseparation and cytotoxicity analysis. J Microbiol Methods 60:259–268CrossRefGoogle Scholar
  24. Gray KM, Banada PP, O’Neal E, Bhunia AK (2005) Rapid Ped-2E9 cell-based cytotoxicity analysis and genotyping of Bacillus species. J Clin Microbiol 43:5865–5872CrossRefGoogle Scholar
  25. 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
  26. Jayme DW, Blackman KE (1985) Culture media for propagation of mammalian cells, viruses, and other biologicals. Adv Biotechnol Process 5:1–30Google Scholar
  27. Kovar J, Franek F (1984) Serum-free medium for hybridoma and parental myeloma cell cultivation: a novel composition of growth-supporting substances. Immunol Lett 7:339–345CrossRefGoogle Scholar
  28. Long WJ, Palombo A, Schofield TL, Emini EA (1988) Effects of culture media on murine hybridomas: definition of optimal conditions for hybridoma viability, cellular proliferation, and antibody production. Hybridoma 7:69–77Google Scholar
  29. Lubiniecki AS (1999) Elimination of serum from cell culture medium. Dev Biol Stand 99:153–156Google Scholar
  30. Lund T, Granum PE (1997) Comparison of biological effect of the two different enterotoxin complexes isolated from three different strains of Bacillus cereus. Microbiology 143:3329–3336CrossRefGoogle Scholar
  31. Lund T, Granum PE (1999) The 105-kDa protein component of Bacillus cereus non-haemolytic enterotoxin (Nhe) is a metalloprotease with gelatinolytic and collagenolytic activity. FEMS Microbiol Lett 178:355–361CrossRefGoogle Scholar
  32. Mascotti K, McCullough J, Burger SR (2000) HPC viability measurement: trypan blue versus acridine orange and propidium iodide. Transfusion 40:693–696CrossRefGoogle Scholar
  33. Menon A, Shroyer ML, Wampler JL, Chawan CB, Bhunia AK (2003) In vitro study of Listeria monocytogenes infection to murine primary and human transformed B cells. Comp Immunol Microbiol Infect Dis 26:157–174CrossRefGoogle Scholar
  34. Miller WM, Blanch HW, Wilke CR (2000) A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: effect of nutrient concentration, dilution rate, and pH (reprinted from Biotechnol Bioeng 32:947–965, 1988). Biotechnol Bioeng 67:853–871CrossRefGoogle Scholar
  35. Ozturk SS, Palsson BO (1991) Growth, metabolic, and antibody production kinetics of hybridoma cell culture: 2. Effects of serum concentration, dissolved oxygen concentration, and medium pH in a batch reactor. Biotechnol Prog 7:481–494CrossRefGoogle Scholar
  36. Pancrazio JJ, Whelan JP, Borkholder DA, Ma W, Stenger DA (1999) Development and application of cell-based biosensors. Ann Biomed Eng 27:697–711CrossRefGoogle Scholar
  37. Pardee AB (1974) A restriction point for control of normal animal cell proliferation. Proc Natl Acad Sci USA71:1286–1290CrossRefGoogle Scholar
  38. Pedersen PB, Bjornvad ME, Rasmussen MD, Petersen JN (2002) Cytotoxic potential of industrial strains of Bacillus sp. Regul Toxicol Pharmacol 36:155–161CrossRefGoogle Scholar
  39. Pine L, Kathariou S, Quinn F, George V, Wenger JD, Weaver RE (1991) Cytopathogenic effects in enterocyte-like Caco-2 cells differentiate virulent from avirulent Listeria strains. J Clin Microbiol 29:990–996Google Scholar
  40. Rawson DM, Willmer AJ, Turner AP (1989) Whole-cell biosensors for environmental monitoring. Biosensors 4:299–311CrossRefGoogle Scholar
  41. Roche SM, Velge P, Bottreau E, Durier C, Marquet-van der Mee N, Pardon P (2001) Assessment of the virulence of Listeria monocytogenes: agreement between a plaque-forming assay with HT-29 cells and infection of immunocompetent mice. Int J Food Microbiol 68:33–44CrossRefGoogle Scholar
  42. Shroyer ML, Bhunia AK (2003) Development of a rapid 1-h fluorescence-based cytotoxicity assay for Listeria species. J Microbiol Methods 55:35–40CrossRefGoogle Scholar
  43. Siddique IH (1969) Cytotoxic activity of hemolysin from Listeria monocytogenes on L-M strain of mouse cells. Can J Microbiol 15:955–957CrossRefGoogle Scholar
  44. Simpson NH, Singh RP, Perani A, Goldenzon C, al-Rubeai M (1998) In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of the bcl-2 gene. Biotechnol Bioeng 59:90–98CrossRefGoogle Scholar
  45. Simpson NH, Singh RP, Emery AN, and al-Rubeai M (1999) Bcl-2 over-expression reduces growth rate and prolongs G1 phase in continuous chemostat cultures of hybridoma cells. Biotechnol Bioeng 64:174–186CrossRefGoogle Scholar
  46. Singh RP, al-Rubeai M (1998) Apoptosis and bioprocess technology. Adv Biochem Eng Biotechnol 62:167–184Google Scholar
  47. Stenger DA, Gross GW, Keefer EW, Shaffer KM, Andreadis JD, Ma W, Pancrazio JJ (2001) Detection of physiologically active compounds using cell-based biosensors. Trends Biotechnol 19:304–309CrossRefGoogle Scholar
  48. Van Langendonck N, Bottreau E, Bailly S, Tabouret M, Marly J, Pardon P, Velge P (1998) Tissue culture assays using Caco-2 cell line differentiate virulent from non-virulent Listeria monocytogenes strains. J Appl Microbiol 85:337–346CrossRefGoogle Scholar
  49. Westbrook DG, Bhunia AK (2000) Dithiothreitol enhances Listeria monocytogenes mediated cell cytotoxicity. Microbiol Immunol 44:431–438Google Scholar
  50. Zanghi JA, Schmelzer AE, Mendoza TP, Knop RH, Miller WM (1999) Bicarbonate concentration and osmolality are key determinants in the inhibition of CHO cell polysialylation under elevated pCO(2) or pH. Biotechnol Bioeng 65:182–191CrossRefGoogle Scholar
  51. Ziegler C (2000) Cell-based biosensors. Fresenius J Anal Chem 366:552–559CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Pratik Banerjee
    • 1
  • Mark T. Morgan
    • 2
  • Jenna L. Rickus
    • 3
  • Kathy Ragheb
    • 4
  • Carlos Corvalan
    • 2
  • J. Paul Robinson
    • 4
  • Arun K. Bhunia
    • 1
  1. 1.Molecular Food Microbiology Laboratory, Department of Food SciencePurdue UniversityWest LafayetteUSA
  2. 2.Department of Food SciencePurdue UniversityWest LafayetteUSA
  3. 3.Department of Agricultural and Biological Engineering and Weldon School of Biomedical EngineeringPurdue UniversityWest LafayetteUSA
  4. 4.Purdue University Cytometry Laboratory, the Bindley Bioscience CenterPurdue UniversityWest LafayetteUSA

Personalised recommendations