, Volume 15, Issue 1, pp 117–128

Induction of apoptosis in oxygen-deprived cultures of hybridoma cells

  • Sylvain Mercille
  • Bernard Massie

DOI: 10.1007/BF00762386

Cite this article as:
Mercille, S. & Massie, B. Cytotechnology (1994) 15: 117. doi:10.1007/BF00762386


It is now well documented that apoptosis represents the prevalent mode of cell death in hybridoma cultures. Apoptotic or programmed cell death occurs spontaneously in late exponential phase of batch cultures. Until lately, no specific triggering factors had been identified. Recently, we observed that glutamine, cystine or glucose deprivation induced apoptosis in both hybridoma and myeloma cell lines whereas accumulation of toxic metabolites induced necrotic cell death in these cells. Other triggering factors such as oxygen deprivation might also be responsible for induction of apoptosis. In the present study, induction of cell death by exposure to anoxia was examined in batch culture of the SP2/0-derived hybridoma D5 clone. The mode of cell death was studied by morphological examination of acridine orange-ethidium bromide stained cells in a 1.5 L bioreactor culture grown under anoxic conditions for 75 hours. Under such conditions, viable cell density levelled off rapidly and remained constant for 25 hours. After 45 hours of anoxia, cell viability had decreased to 30% and the dead cell population was found to be 90% apoptotic. In terms of cellular metabolism, anoxia resulted in an increase in the utilization rates of glucose and arginine, and in a decrease in the utilization rate of glutamine. The lactate production rate and the yield of lactate on glucose increased significantly while the MAb production rate decreased. These results demonstrate that glycolysis becomes the main source of energy under anoxic conditions.

Cells incubated for 10 hours or less under anoxic conditions were able to recuperate almost immediately and displayed normal growth rates when reincubated in oxic conditions whereas cells incubated for 22 hours or more displayed reduced growth rates. Nonetheless, even after 22 h or 29 h of anoxia, cells reincubated in oxic conditions showed no further progression into apoptosis. Therefore, upon removal of the triggering signal, induction of apoptosis ceased.

Key words

Animal cell culture anoxia apoptosis cell death hybridoma hypoxia 



Viable non-apoptotic cells


Viable apoptotic cells


Nonviable non-apoptotic or necrotic cells


Nonviable apoptotic cells


Chromatin-free cells (late nonviable apoptotic cells)


Acridine orange


Ethidium Bromide


Monoclocnal antibody


Dissolved oxygen


Specific MAb production rate (mg. (109 cells)−−1)


Specific growth rate (h−1)


Viable cell number (105 cells.mL−1)


Total cell number (105 cells.mL−1)


Yield coefficient of lactate on glucose (mM lactate produced/mM glucose consumed)

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Sylvain Mercille
    • 1
  • Bernard Massie
    • 2
  1. 1.Biomira Inc.MontréalCanada
  2. 2.Groupe d'Ingénierie des cellules animales, Institut de recherche en BiotechnologieConseil National de Recherches du CanadaMontréalCanada

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