True Hypoxia Replication: Creating Optimal Conditions for Cell-Based Research

There is a rapidly growing need for systems capable of creating precise, reproducible and physiologically relevant cell environments in the laboratory in order to enable accurate analysis of both cell metabolism and cell function. Today’s investigators and researchers now recognize that the far-reaching impact of unnatural cellular environments, such as exposing cultures to room air oxygen levels, includes the risk of erroneous outcomes and distorted research outcomes.

Figure 1 – HypoxyLab hypoxia workstation.

New-generation hypoxia workstations, such as the HypoxyLab™ from Oxford Optronix (Abingdon, U.K.) (see Figure 1), make it possible for research teams to accurately reproduce real-life physiological conditions for a wide variety of cell-based research fields, including cancer biology, radiation cell biology, cardiovascular research, apoptosis, neurology, stem cell research, 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. While in the past the focus in the laboratory was on maintaining cells alive and genetically stable while avoiding contamination, in recent decades researchers have demonstrated that cells react in different ways, both metabolically and morphologically, depending on the environmental factors that maintain and interact with them.

The impact of oxygen

Atmospheric oxygen concentrations in classic open in vitro cultures are far higher than those experienced by in vivo cells, where oxygen levels are much lower, and this difference has far-reaching consequences. Studies have shown that exposing cells to air containing 21% oxygen levels can trigger cellular stress, invoking significant physiological changes that include differentiation, growth factor signaling and other cellular processes.

As cell culturing advances and new technologies such as cell-based human therapies emerge, the need for systems that can generate reproducible, precise and physiologically relevant cell environments is becoming ever more critical. Today’s 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.

In the field of cancer research, for example, the hypoxia-inducible factor (HIF) in cancer cells illustrates the potential for specific oxygen tensions to dramatically affect the post-translational modification of proteins. As a tumor 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.

As a result, in today’s cancer research, scientists are looking to recreate in the laboratory (in vitro) the same conditions as found in the body (in vivo) in order to study tumor mechanisms and perform candidate drug testing under relevant and reproducible conditions.

Investigating the effects of hypoxia

In recent decades there has been a growing interest in the role of low oxygen, or hypoxic, conditions with regard to the behavior mechanisms of tumor cells. Hypoxic tumor regions often produce the most aggressive and therapy-resistant cells, causing tumors to spread. Understanding how cell behavior is affected by hypoxia has led to researchers undertaking investigations on how to prevent hypoxia-mediated metabolic changes, with the aim of making tumor cells more responsive to current cancer treatments. The ability to manage environmental parameters such as oxygen and carbon dioxide concentrations, temperature and humidity is critical to the ability of research teams to recreate the requisite low oxygen conditions in vitro and undertake accurate analysis of the metabolic transformation process cancer cells undergo in order to survive and grow.

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 also growing as to 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 also being implicated in the pathogenesis of certain benign diseases, 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.

Creating the right research 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, yet much cell biology and research is still being undertaken in traditional incubators that expose cells to normal atmospheric oxygen levels.

Today’s life science and clinical/medical researchers need specialized hypoxic workstations that deliver the ability to produce customized in vivo environments in which cells can be cultured 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.

Connected to gas supplies to support purging with nitrogen, 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 laboratories need to assure hypoxia replication precision, 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 expressed hypoxia in terms of barometrically uncompensated percent oxygen concentration alone, this scientifically superior approach characterizes the chamber environment using the partial pressure of oxygen expressed in units of mmHg or kPa, and is insensitive to changing climatic conditions or the altitude of the research location.

Since the partial pressure of oxygen—what cells actually “see” when exposed to oxygen— not only with oxygen concentration in the atmosphere, but also with altitude and prevailing weather conditions, this revolutionary 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 setpoints. This ensures that, regardless of whether research laboratories 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 nebulizer-based humidifier technology. Using touchscreen displays, operators also have the ability to set the hypoxia workstations to automatically cycle through up to eight fully programmable profiles.

Direct oxygen measurement from cell culture media

The measurement of respiration rates is a versatile tool for the diagnosis of cellular and metabolic state and the ability to directly determine the oxygen update rate of cellular samples has broad implications in furthering the understanding of a wide range of biological systems.

Today’s second-generation hypoxia workstations further extend the boundaries of research capability by enabling “gold standard” in situ oxygen measurements from the precise location or layer in which cells are growing.

By positioning an additional sensor within the culture, researchers are able to gain precise dissolved oxygen measurements direct from within the cell-culture media itself, alongside real-time values of chamber pO2, pCO2, temperature and humidity.

This means that operators can directly measure the oxygen concentration cells are actually exposed to. This unique additional research dimension gives scientists the ability to compensate for metabolic oxygen consumption by the cell culture.

Accurate, reproducible and physiological cell environments

Today’s next-generation “contamination-free” workstations are making it possible for researchers to accurately reproduce real-life physiological conditions for a wide variety of cell-based research fields, including cancer biology, cardiovascular research, apoptosis, virology, neurology, stem cell research, multidisciplinary drug development and proteomics.

By delivering true hypoxia replication, using oxygen partial pressure in a chamber that is insensitive to changing climatic and altitude conditions, these workstations also extend the research capabilities of laboratories yet further by making it possible to capture real-time dissolved oxygen measurements directly from the cell-culture media itself.

For research teams undertaking studies at the cellular and molecular level, hypoxia workstations that enable accurate hypoxia replication are proving essential to refine the processes they are using to probe gene expression changes, signal transduction pathways, enzyme activities and the molecular mechanisms that underlie these phenomena.

Michael Rau, Ph.D., is Director for Sales and Marketing at Oxford Optronix, 19-21 East Central, 127 Olympic Ave., Milton Park, Abingdon OX14 4SA, U.K.; tel.: +44 1235 821 803; e-mail: [email protected];  www.oxford-optronix.com

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