Cell death in bioreactors: A role for apoptosis

Abstract

The incidence of apoptotic and necrotic cell death was compared in CHO, SF9 insect cells and murine plasmacytoma (J558L) and hybridoma (TB/C3) cells during in vitro cultivation in batch cultures. Acridine orange staining and fluorescence microscopy enabled the visualization of a classic morphological feature of apoptotic cell, the presence of condensed and/or fragmented chromatin. DNA gel electrophoresis was employed to show an additional characteristic of the process, the endonuclease-mediated fragmentation of DNA into multiples of 180 base pairs. The levels of apoptosis at the end of batch cultures of plasmacytoma and hybridoma cell lines were found to be 60% and 90% of total dead cells, respectively. However, employing the above-mentioned techniques, the biochemical and morphological features of apoptosis were not found in CHO and SF9 insect cells. Some factors affecting the induction of apoptosis during the batch culture of the hybridoma and plasmacytoma cell lines were identified. The most effective inducer was found to be glutamine limitation, followed by (in order of importance) serum limitation, glucose limitation, and ammonia toxicity. Blockage of the cell cycle of the plasmacytoma and hybridoma cells using thymidine resulted in the induction of apoptosis. This has important implications for the development of cell culture processes that minimize cell division and thereby increase specific productivity.

Apoptosis and Its Suppression in Hepatocytes Culture

“…Amino acids considered essential for cellular proliferation were not always revealed as essential for survival, in particular glutamine. This discovery was in stark contrast to reports describing the induction of apoptosis in the absence of glutamine from the culture of many cell lines (Mecille and Massie 1994;Singh et al 1994;Simpson et al 1998;Sanfelui and Stephanopoulos 1999). Only cells that endogenously express GS synthetase or those genetically engineered to do so would be expected to survive such conditions as they are able to convert glutamic acid and ammonia to glutamine.…”

“…However, in the present study no evidence of apoptosis was observed by fluorescence microscopy, in fact ammonia levels studied did not have a detrimental affect on either HepZ proliferation or survival. During HepZ batch culture, the cumulative ammonia levels recorded exceeded cytotoxic levels in hybridoma and myeloma cultures, with a concentration reaching 7.4 mM recorded (not shown) (Mecille and Massie 1994;Singh et al 1994;Tey et al 2000b). No adverse affects were observed on increasing ammonia levels to 14 mM, indicating that HepZ have retained the ability to metabolise excess ammonia via ureogenesis.…”

“…Although early interest in apoptosis stemmed from cancer research, the discovery of apoptosis in hybridoma cultures highlighted the significance of apoptosis to the biotechnology industry (Al-Rubeai et al 1990). Within the bioreactor environment apoptosis can be triggered by a number of stimuli, including exhaustion of essential nutrients (amino acids, serum components, and glucose), accumulation of metabolic by-products, and alterations in pH, dissolved oxygen content and medium osmolarity (Singh et al 1994;Simpson et al 1998;Al-Rubeai 1999, 2002). Additional environmental factors that lead to apoptotic cell death in large-scale mammalian cell cultures include those caused by mechanical stresses (Al-Rubeai et al 1995).…”

Inhibitory effects of persistent apoptotic cells on monoclonal antibody production in vitro

“…Cell death through apoptosis in cultures of antibodyproducing hybridoma cells is well known, 31,32 and strategies for improving viability of hybridoma cells via suppression of their apoptosis program (such as through expression of Bcl-2-family proteins) to improve antibody productivity have been widely studied. 33,34 However, to our knowledge, the phenomenon described here of substantially improved antibody production by hybridoma cells following removal of non-viable cells prior to culture seeding has not been reported previously.…”

Dynamic Modeling of Apoptosis and its Interaction with Cell Growth in Mammalian Cell Culture

“…This is caused when the cells are exposed to sudden and harsh cellular damage occurring upon severe changes in culture conditions such as shear stress on cell membrane due to collision with the bioreactor wall or stirrer, exposure to high levels of toxic metabolites (Singh et al, 1994).…”