Dealing with cell death

Apoptosis is a major cause of cell death in live cell in vitro scientific processes, yet little is done to separate out dead or dying cells, which can dramatically affect the productivity and viability of cellular systems. A revolutionary new technology is set to change the way we manage apoptosis in cell cultures, offering innovative methods for recognising and removing apoptotic and necrotic cells, using simple magnetic extraction techniques. This article discusses the potential benefits of extracting apoptotic cell detritus from live cells, across a range of research and commercial processes.

APOPTOSIS is the process of programmed cell death which occurs in a wide range of multicellular organisms. Unlike necrosis due to cell trauma, this process is characterised by a distinct series of biochemical events which results in the eventual destruction of the cell. In mammals, the immune system is able to identify these dead or dying cells through their unique surface morphology, leading to phagocytosis and removal of apoptotic cell material from the body. This process is essential for maintenance of healthy tissues, promoting new cell growth and preventing inflammatory responses.

By contrast, in vitro cell cultures have no mechanism for the removal of apoptotic cells despite the fact that the behaviour of a vast majority of cell types is directly influenced by their immediate environment due to a wide array of receptors on the exterior of the cell membrane. It is no surprise, therefore, that the presence of debris from dead and dying cells in the media of suspension cell cultures has a significant effect on the behaviour of neighbouring viable cells. The existence of such cells within cell cultures is an inevitable result of normal cellular activity, yet little is currently done to remove apoptotic and necrotic material. Study of in vivo cellular processes indicates that the presence of dead cells adversely affects the growth and metabolic activity of viable cells. This cellular debris influences the efficiency of molecular processes, and can be a source of non-specific background ‘noise’ in a wide range of cellular experiments and assays. For example, enzymatic digestion of nucleic acids occurs rapidly upon cell death, and the presence of degraded RNA is highly problematic to techniques such as array gene expression analysis. Similarly, chemotactic migration of cells is highly dependent on chemical sensing by receptors on the surface of viable cells, and various factors released by dying cells influence the migration of neighbouring cells. To avoid the negative impact of dead cells on in vitro cultures, a simple mechanism is needed to remove this material and improve the efficiency of cell culture processes. Although there have been several attempts to develop methods for removal of dead cells in the past, these techniques have involved unfavourable or harsh culture handling steps, causing the death of many healthy cells.

In 2004, researchers in the School of Medicine and Veterinary Medicine at the University of Edinburgh began investigating new methods for removal of apoptotic material, using antibody technology to recognise distinct biomarkers on the exterior surface of cell membranes. This strategy exploits the cell membrane’s characteristic phospholipid asymmetry, which is lost during the early phase of apoptosis, causing phospholipids and other molecules normally only present on the inner leaflet of the membrane to become visible extracellularly. The exposure of anionic phospholipids, such as phosphatidylserine (PS), offers a reliable method for identification of apoptotic cells at a far earlier stage of cell death than existing methods, which rely on disruption of the cellular membrane.

Figure1: Experimental set-up for chemotactic migration of neutrophils towards fMLP in the lower compartment of a Boyden chamber.

To exploit this change in membrane topology, the group in Edinburgh developed a mouse monoclonal antibody, imab6, which binds with high affinity to PS. Although several other known PS-binding proteins have previously been used for bio-recognition of apoptotic cells, these proteins require unfavourable conditions to allow high affinity binding. For example, annexin V – which has a role in inhibiting coagulation of blood – is used to monitor apoptosis by flow cytometry. However, this requires high Ca2+ concentrations to enable effective binding, requiring transfer of cells to a non-optimal high Ca2+ medium for analysis, leading to cell clumping and further cell death. Using imab6 allows faster, more efficient identification of apoptotic cells in vitro, without the need for adverse manipulation of cell cultures. Necrotic cells and cell debris can also be detected using this antibody - due to large scale disruptions of the cell membrane exposing PS molecules - offering robust identification of all dead or dying cells, regardless of the mechanism of cell death.

This straightforward system offers a valuable tool for researchers investigating apoptotic processes or merely requiring proper assessment of viability, and has potential applications in a wide variety of cell culture systems for identification and removal of dead and dying cells. Flow cytometry is one example of this, using imab6 antibodies for direct recognition of apoptotic cells. However, it is use of this technology for removal of dead cells from live cultures which offers the greatest benefits. A variety of immobilisation strategies have been investigated to allow the dead cell depletion using imab6, including a wide range of solid supports, however the majority of these require undesirable manipulation of the culture, and so have met with limited success.

Immobilisation of imab6 antibodies onto super-paramagnetic nanoparticles offers a straightforward and minimally invasive mechanism for removal of dead and dying cells from live culture, ensuring good cell viability rates (Figure 1). Antibody-laden particles can be added to the cell culture under optimal conditions and, following incubation, these particles will be immobilised onto the surface of apoptotic cells and necrotic cell debris. The large size of cells relative to the particles results in many particles binding to each apoptotic cell, allowing these cells to be easily separated out from viable cells using magnetic separation techniques. This system offers simple and effective removal of dead and dying cells from live cell cultures without adverse culture manipulation. Furthermore, the use of a proprietary polymer on the particle surface provides additional discriminatory capacity in selectively binding a very wide variety of non-viable cells. Now commercialised by ImmunoSolv as Dead-Cert Nanoparticles, this groundbreaking technology is distributed in the UK by Flowgen Bioscience, the life science division of Scientific Laboratory Supplies

One of the most common causes of cell death in laboratory research is cryo-damage due to repeated freeze-thaw cycles, with even a single cycle of freezing and thawing leading to a large drop in the number of viable cells remaining in culture. Though poorly understood, the inhibitory effect caused by the presence of dead cells can severely impact on the activity of viable cells, and removal of apoptotic and necrotic cells significantly improves the biosynthesis efficiency of cultures. For monoclonal antibody production using hybridoma cells, using Dead-Cert Nanoparticles to remove dead cells immediately prior to incubation can improve yield by over 100%. This straightforward culture clean-up process potentially offers large savings in time, labour and resources, driving down the cost of bioproduction processes.

In addition to improving yield for biomanufacturing processes, dead cell depletion of cell cultures actually improves the growth rates of viable cells. With the rapidly growing interest in stem cells for use in therapeutics, this system potentially offers substantial advantages for cell growth, improving growth rates and minimising potential contamination during culture re-seeding. For example, growth of E14-IV murine stem cells can be improved by over 50% using imab6 nanoparticles to remove dead cells at the time of passage. The application of the technology is not limited to cultured cell lines; any population of cells, including freshly isolated primary cells, can be improved using Dead-Cert Nanoparticles.

Removal of dead and dying cells from culture prior to processing can significantly improve the quality of results for downstream assays. Use of Dead-Cert Nanoparticles in preparation of RNA from human monocytic leukemia cells eliminates non-specific interference from species of degraded RNA, simplifying purification and analysis, and improving results. This strategy can also be used to enhance cell sensing processes such as chemotaxis, as the various factors released by apoptotic and necrotic cells have a strong influence the migration of neighbouring viable cells. Classical cell migration experiments in a Boyden chamber – testing the ability of cells to migrate toward a chemo-attractant – demonstrate marked improvements in the migration ability of viable cells following removal of dead and dying cells. Other applications include improving drug selection and transfection efficiencies.

The presence of dead and dying cells in in vitro cultures has a negative impact on the growth and activity of neighbouring viable cells. The signalling pathways and exact mechanisms by which cell corpses affects viable cells in culture have not yet been fully explored, yet the benefits of removing this material from cell cultures are now beginning to be recognised. Historically, attempts to remove dead cell material have been impractical and inefficient, making cell death and the resulting drop in culture efficiency an unfortunate yet unavoidable consequence of in vitro culture processes. Removal of apoptotic and necrotic cells is now straightforward and practical using magnetic Dead-Cert Nanoparticles, offering a simple method to improve the results of cell-based assays and enhance biomanufacturing for a wide range of applications.