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European Biopharmaceutical Review

Prime Conditions

There is widespread recognition that the ability to create physiologically reproducible, low-oxygen and hypoxic environments for mammalian cells in the laboratory is vital for the accurate analysis of both cell metabolism and cell function. A new generation of hypoxia workstations is making it possible for research teams to accurately replicate real-life physiological conditions for a variety of cell-based research fields – including cancer biology, radiation cell biology, cardiovascular treatments, apoptosis, neurology, stem cell therapy, multidisciplinary drug development and proteomics.

Interest in the role of hypoxia in cell development has gained momentum as researchers learn more about the biology of physiological oxygen concentration and how it affects cells at the molecular level. In recent decades, researchers have demonstrated that cells react in different ways, both metabolically and morphologically, depending on the environmental factors maintaining and interacting with them.

This is because the body maintains cellular oxygen concentrations within a narrow range, due to the risk of damage from too much oxygen and death from too little oxygen. The ‘normal’ amount of oxygen for a cell depends on where that cell is in the body and what it does. Therefore, regulating oxygen levels for cell cultures takes researchers a step closer to what goes on in real life.

Perfect Conditions

Within the body, oxygen concentrations typically range from 1-12%, rather than the 21% in the atmosphere. Studies conducted in the past decade have revealed that exposing cells to unnaturally rich oxygen environments, even for a short time, can trigger cellular stress and invoke significant physiological changes such as differentiation, growth factor signalling, and other cellular processes including post-translational metabolic pathways.

As cell culturing advances and new technologies like cell-based human therapies emerge, the need for systems capable of creating precise, reproducible and physiologically relevant cell environments is becoming ever-more critical. Researchers need to be able to mimic the conditions in the body where cells originate, accurately simulating physiological oxygen concentrations and duplicating other environmental factors, such as carbon dioxide, temperature and humidity levels.

For example, in the field of oncology research, the hypoxiainducible factor (HIF) in cancer cells illustrates the potential for specific oxygen tensions to dramatically affect the posttranslational modification of proteins. As a tumour grows, its oxygen levels start to fall, triggering the HIF-1 protein response which enables the cancer cells to adapt to scarce oxygen levels and continue to grow. Therefore, in today’s cancer research, scientists are looking to recreate in the laboratory the same conditions found in the body, in order to study tumour mechanisms and perform candidate drug testing under relevant and reproducible conditions.

Effects of Hypoxia

Research on the effects of hypoxia has blossomed in recent years with the realisation that it modulates a variety of normal developmental and metabolic processes. This interest has its roots in research that first began with radiation biologics and oncologists in the 1900s, alongside a growing understanding of how hypoxia increases tumour resistance to radiation therapy. By the 1950s, researchers operating in this field had demonstrated that the oxygen-deprived microenvironment in human tumours plays a key role in the pathogenesis and progression of cancer, and that decreased oxygenation leads to various biochemical responses in tumour cells. By the 1990s, the importance of oxygen in determining the outcome of human tumour growth and cell survival end-points was well-established, and the role of HIF was emerging.

Since those early days, the role of hypoxia in cancer research has broadened considerably. Tumour hypoxia has been shown to be a marker of disease progression and a key influencer in the outcome of chemotherapy and radiotherapy. Hypoxic tumour cells have indicated that they are resistant to radiation and many anti-cancer drugs, and approaches to circumventing the effects of hypoxia are now being examined – new agents currently being tested in preclinical studies and clinical trials offer the possibility that better hypoxia-directed and novel therapies will become available in the near future.

It is also becoming clear that hypoxia is important during embryonic development, in the physiology of certain normal tissues, and in the maintenance of the phenotypes of certain stem cells. Recognition is growing of the important role oxygen plays in maintaining stem cell fate in terms of self-proliferation and differentiation, as well as the sequential steps that follow engraftment.

Chronic moderate hypoxia is being implicated in the pathogenesis of certain benign diseases too, including some retinopathies and complications of diabetes. Similarly, virology laboratories are also engaged in investigating the relevance of low oxygen environments in relation to viral infection.

The Right Environment

In the last decade, researchers have demonstrated that culturing different cell types in low oxygen environments that are ‘normal’ for cells in the body generates more biologically relevant results. However, the majority of cell biology and research is still undertaken in traditional incubators which expose cells to atmospheric oxygen levels. As cell culture progresses, the standard incubator is no longer adequate for producing the customised in vivo environments and conditions required for life sciences and clinical medical research applications.

To accurately reproduce the relevant physiological conditions required for current cell-based research, a contaminationfree working environment is required – one that offers the continuous control of oxygen, carbon dioxide gas, temperature and humidity, and enables cells in vivo to be maintained in their natural state at oxygen concentrations in the range of 0.5-10%, depending on tissue type.

The first generation of hypoxia workstations were designed to supply independent oxygen and carbon dioxide control and monitoring, together with humidification, to enable cell biology research to be performed over a range of oxygen tensions. Gas supplies are connected to support purging with nitrogen, while oxygen levels are simply controlled and presented to users as a percentage measurement. However, this approach does not take account of barometric or atmospheric changes, and risks failing to deliver the pinpoint accuracy research labs need in order to ensure precise hypoxia replication, regardless of the altitude of the research location or the ambient climatic conditions.

True Hypoxia Replication

Today’s second-generation and precision-controlled hypoxia workstations use the absolute partial pressure of oxygen in the chamber to deliver authentic hypoxia replication with the highest possible accuracy.

Unlike first-generation workstations – which simply express hypoxia in terms of a barometrically uncompensated percentage of oxygen concentration alone – this approach characterises the chamber environment using the partial pressure of oxygen, expressed in units of millimetres of mercury (mmHg) or kilopascals (kPa), and is insensitive to changing climatic conditions or the altitude of the research location.

Since the partial pressure of oxygen – which is what cells actually ‘see’ when exposed to oxygen – can vary not only with oxygen concentration in the atmosphere, but also with altitude and prevailing weather conditions, this technological advancement enhances hypoxia accuracy by as much as 30% over first-generation devices, and is of particular value when culturing cells at extremely low oxygen set points. This ensures that, regardless of whether research labs are located in Amsterdam or Denver, truly accurate hypoxia profiles can be configured and generated reliably.

Full control of the partial pressures of oxygen and carbon dioxide, alongside chamber temperature and humidity, is delivered using digital electronic gas flow controllers, autocalibrating sensors and built-in nebuliser-based humidifier technology. Using touch-screen displays, operators also have the ability to set the hypoxia workstations to automatically cycle through up to eight fully programmable profiles.

Gold Standard Measurement

The second-generation hypoxia workstations further extend the boundaries of research capability through the provision of a facility that makes it possible to support ‘gold standard’ in situ oxygen measurements from the precise location or layer in which cells are growing. A built-in module allows an additional sensor to be positioned within the culture and deliver dissolved oxygen measurements direct from within the cell-culture media itself. This enables operators to directly measure the oxygen concentration that cells are actually exposed to, giving scientists the ability to compensate for metabolic oxygen consumption by the cell culture. Values representing the precise dissolved oxygen concentration available to cells under culture are automatically displayed and recorded, along with the values of chamber pO2, pCO2, temperature and humidity.

Physiological Cell Environments

Next-generation, contamination-free workstations allow scientists to reproduce the exact physiological conditions needed to research many cell-based fields, with greater accuracy than ever before.

Delivering true hypoxia replication using oxygen partial pressure in a chamber that is insensitive to changing climatic and altitude conditions, these workstations further extend the boundaries of research capabilities by capturing realtime dissolved oxygen measurements directly from the cell-culture media.

While earlier studies documented the effects of hypoxia on inhibiting cell proliferation and certain DNA repair pathways, altering glucose metabolism and energy balance, cellular redox status and drug metabolism, current studies at the cellular and molecular level are probing gene expression changes, signal transduction pathways, enzyme activities and the molecular mechanisms that underlie these phenomena. Research teams are embracing a new era of hypoxia workstations that enable truly accurate hypoxia replication.

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Michael Rau PhD is Director for Sales and Marketing at Oxford Optronix. Following a doctorate on the molecular biology of mammalian cell gene expression at the University of Sussex, he initially pursued an academic pathway with a three-year post at a human virology research lab in Lyon. Michael joined Oxford Optronix as a Product Specialist in 1999, following the launch of the market’s first dedicated fluorescence lifetime-based, fibre-optic tissue oxygen monitor, the OxyLite™. He has an in-depth technical understanding of the company’s products and their application.
Michael Rau
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