Hybridoma Ped-2E9 cells cultured under modified conditions can sensitively detect Listeria monocytogenes and Bacillus cereus
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.
KeywordsPed-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.
- 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
- 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
- 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
- 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
- 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
- Froud SJ (1999) The development, benefits and disadvantages of serum-free media. Dev Biol Stand 99:157–166Google Scholar
- Jayme DW, Blackman KE (1985) Culture media for propagation of mammalian cells, viruses, and other biologicals. Adv Biotechnol Process 5:1–30Google Scholar
- 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
- Lubiniecki AS (1999) Elimination of serum from cell culture medium. Dev Biol Stand 99:153–156Google Scholar
- 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
- Singh RP, al-Rubeai M (1998) Apoptosis and bioprocess technology. Adv Biochem Eng Biotechnol 62:167–184Google Scholar
- Westbrook DG, Bhunia AK (2000) Dithiothreitol enhances Listeria monocytogenes mediated cell cytotoxicity. Microbiol Immunol 44:431–438Google Scholar