Life and death in mammalian cell culture: strategies for apoptosis inhibition

doi:10.1016/j.tibtech.2004.02.004

Trends in Biotechnology

Volume 22, Issue 4, April 2004, Pages 174-180

https://doi.org/10.1016/j.tibtech.2004.02.004 Get rights and content

Abstract

Mammalian cell culture is widely used to produce valuable biotherapeutics including monoclonal antibodies, vaccines and growth factors. Industrial cell lines such as Chinese hamster ovary (CHO), mouse myeloma (NS0), baby hamster kidney (BHK) and human embryonic kidney (HEK)-293 retain many molecular components of the apoptosis cascade. Consequently, these cells often undergo programmed cell death upon exposure to stresses encountered in bioreactors. The implementation of strategies to control apoptosis and enhance culture productivities represents a major goal of biotechnologists. Fortunately, previous research has uncovered many intracellular proteins involved in activating and inhibiting apoptosis. Here, we summarize three apoptotic pathways and discuss different environmental and genetic methodologies implemented to limit cell death for biotechnology applications.

Section snippets

Cell death by apoptosis

Apoptosis is a genetically controlled process and is morphologically recognized by cell and chromatin shrinkage followed by plasma membrane blebbing. Blebbing involves the shedding of membrane fragments from the cell in the form of apoptotic bodies that often include cytosolic and nuclear contents. An apoptotic Chinese hamster ovary (CHO) cell exhibiting membrane blebbing and chromatin shrinkage is compared to a wild-type CHO cell following staining with acridine orange and ethidium bromide (

Apoptotic detection

Assays to detect apoptosis in cell populations include measuring DNA fragmentation with DNA ladders, a signature of apoptosis, or detecting activation of apoptosis-induced proteases, such as caspase-3 or poly(ADP ribose) polymerase (PARP) using Western blot techniques. Methods for detecting apoptosis in individual cells include terminal deoxynucleotidyl transferase nick-end-labeling (TUNEL), annexin V binding to cell membranes and DNA staining with propidium iodide, 4,6-diamino-2-phenylindole

Methods for apoptosis inhibition

Given that the induction of apoptosis can lead to the loss of viable cells in bioreactors, several methods are being evaluated to limit the activation of the apoptosis cascade. Inhibiting or slowing the onset of cell death is beneficial because extending cell lifetimes can lead to more-productive cell culture systems for biotechnology applications [12]. Two strategies being examined for enhancing cell survival in bioreactors are the manipulation of the external environment through media

Media supplementation

Given that the onset of apoptosis is often triggered by conditions outside cells, one strategy to limit cell death is to alter the extracellular environment. Altering the media can be a highly effective technique in prolonging cell viability in culture through the addition of nutrients or supplementation with anti-apoptotic chemicals or peptides. Serum is often an effective anti-apoptosis agent during all stages of cell growth following nutrient depletion. However, the growth of cultures in

Genetic strategies

Recent genetic strategies have proven successful in delaying apoptosis in cell culture. Several viral and cellular proteins inhibit apoptosis in cells at distinct points along the apoptotic pathways, and the expression of genes encoding these proteins can often modify the cell-death response in mammalian cell cultures.

Bcl-2 and Bcl-x_L are prominent anti-apoptotic proteins that inhibit the release of pro-apoptotic molecules from the mitochondria. NS0, CHO, BHK and hybridoma cells transfected

Conclusions and future work

The intracellular components of the apoptosis cascade are now being unraveled to reveal a wide array of cellular factors and complex pathways converging in programmed cell death. As we gain better insights into the molecular mechanisms behind this cascade, better strategies will be devised for controlling the cell-death response for animal cells in culture. For example, conditions in the ER are now recognized to have an important role in the onset of apoptosis for some stimuli, and this pathway

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Trends in Biotechnology

Life and death in mammalian cell culture: strategies for apoptosis inhibition

Abstract

Section snippets

Cell death by apoptosis

Apoptotic detection

Methods for apoptosis inhibition

Media supplementation

Genetic strategies

Conclusions and future work

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Curr. Opin. Cell Biol.

Trends Biotechnol.

J. Biotechnol.

J. Biotechnol.

Metab. Eng.

J. Biotechnol.

J. Biotechnol.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

The role of the bcl-2 family in the regulation of outer mitochondrial membrane permeability

Cell Death Differ.

Pro-apoptotic activity of transiently expressed bcl-2 occurs independent of BAX and BAK

J. Cell. Biochem.

Bax-independent inhibition of apoptosis by Bcl-xL

Nature

Apoptosis in batch cultures of Chinese hamster ovary

Biotechnol. Bioeng.

Endoplasmic reticulum stress-mediated apoptosis in pancreatic β-cells

Apoptosis

Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

J. Cell Biol.

Organelle-specific initiation of cell death pathways

Nat. Cell Biol.

Targeting death and decoy receptors of the tumour-necrosis factor superfamily

Nat. Rev. Cancer

The growth factor inhibitor suramin reduces apoptosis and cell aggregation in protein-free CHO cell Batch cultures

Biotechnol. Prog.

Insulin-like growth factor-I and transferrin mediate growth and survival of Chinese Hamster Ovary Cells

Biotechnol. Prog.

Effect of glutamine limitation on the death of attached Chinese hamster ovary cells

Biotechnol. Bioeng.

Selected amino acids protect hybridoma and CHO cells from elevated carbon dioxide and osmolality

Biotechnol. Bioeng.

Bax-independent inhibition of apoptosis by Bcl-x_L