Exposing cells native to low-oxygen (2-8%) environments to normal atmosphere (21% O2) causes abnormal cell interactions and reduces cell viability. Moreover, a controlled environment for achieving and maintaining atmosphere and media conditions within nontoxic pH parameters (7.0-7.4) is crucial for cellular-based work. Therefore, gas controlled incubators and controlled atmosphere workstations are often used to simulate normal conditions in the body’s organ systems and provide a better understanding of understanding of some of the complex processes involved in low oxygen culture work.
While gas controlled incubators and workstations help to control the atmosphere under which research is performed, cell and tissue cultures still remain prey to oxidative stress due to contrasting concentrations of dissolved oxygen between the cells and cell growth media. In fact, new cultured media contains approximately 10-12% dissolved oxygen content by mass, whereas certain tissue cells are typically plated best at 1-3% oxygen, depending upon the research application. To combat this, most researchers are performing their own methods to condition media to a desired level of oxygen concentrations for their research application. Little documented research is available that further examines these methods, including the precision and accuracy of the actual dissolved oxygen content within the cultured media.
When hypoxic conditions are required, the typical process for conditioning (i.e., reducing the oxygen concentration in) cell culture media is time-consuming and unpredictable. A controlled-oxygen chamber is set to the desired oxygen level, while media is incubated in the chamber for some time before inoculating cells. There are three problems with this empirical conditioning approach.
Current practices assume that the dissolved oxygen content in the media will eventually equilibrate the to the desired level of oxygen required for the application. Based on Baker Ruskinn’s research, while the oxygen setting in the chamber may provide 2% oxygen within the environment of the chamber, the actual level of oxygen within the media is much greater and may not reach that level for a significant period of time.
Obtaining true, physiologically relevant oxygen levels in media requires significant time and resources within the laboratory. Figure 2 highlights that – dependent upon the conditions and solutions being utilized – it may take over 2 days to achieve an actual dissolved oxygen concentration of 2% within the media itself. Moreover, this process would need to be repeated each time media conditioning is required.
Baker Ruskinn’s research shows that there are a number of variables that may impact the traditional conditioning process. Depending upon the duration of the conditioning process, we found that pH levels within the media varied, was often skewed to an abnormally high alkaline or low acidity reading. More importantly, the media (which is clean, but not sterile) when capped or HEPA filtered in a bench, workstation or chamber may become susceptible to microbial contamination when conditioned for a significant period of time. Additionally, the conditioning process may also be impacted by other variables such as altitude, temperature and gas exchange (carbon dioxide).
Even if media integrity is preserved, tissue culture media conditioned by traditional methods may contain higher-than-expected dissolved oxygen levels. This may cause cellular behavior reflective of adaption to abnormal oxygen concentrations, rather than a study that reflects a physiologically-accurate oxygen level for that cell-based experiment.
HypoxyCOOL is the first commercialized solution designed to quickly and precisely condition liquid media from an ambient (or unknown) oxygen concentration to a user-defined oxygen level. HypoxyCOOL helps researchers three ways.
HypoxyCOOL helps improve experimental control by eliminating errors in media conditioning. Many variables impact the rate of deoxygenation of liquid media, including but not limited to the type of media being utilized, the temperature range, and your laboratory’s altitude. HypoxyCOOL helps you control these variables and provides a repeatable conditioning protocol that improves the consistency of your results. HypoxyCOOL also helps preserve the integrity of your media during the conditioning cycle, keeping your cells safe from contaminants and variations in pH.
HypoxyCOOL is a vital step for improving cell yield and reducing artifact-driven gene expression changes in all tissue culture processes. Culture media conditioned with HypoxyCOOL boosts the effects of a conventional tri-gas or CO2/O2 incubator or controlled-oxygen workstation by providing a physiologically-accurate oxygen level in the immediate cellular environment, further improving your research results.
Research conducted in Baker Ruskinn’s laboratory and performed independently has shown that using in vivo-like oxygen levels in culture media stabilizes cell cultures, enhances gene expression, while increasing viability and transcriptional stability. Independent research indicates that HypoxyCOOL provides up to 150% reduction in high-oxygen artifact-driven gene expression changes when HypoxyCOOL is used in conjunction with the Ruskinn SCI-tive hypoxia workstation.
Save time and increase productivity in your lab by switching from a conventional media conditioning method to HypoxyCOOL. Conditioning your media with HypoxyCOOL significantly reduces the time required to achieve a desired level of oxygen concentration in the media. A standard HypoxyCOOL cycle (1% O2, 5% CO2, 120 RPM) yields 2% O2 within 2 hours. Additionally, oxygen levels in media conditioned with HypoxyCOOL stay low for up to 21 days, eliminating the need for re-conditioning. Because HypoxyCOOL is fully compatible with tri-gas incubators and hypoxia workstations, it easily integrates with your existing laboratory setup.
Global Sales Director Samir Patel and Science Director Deborah Thibodeaux, Ph.D., presented a poster at the Society for Free Radical Biology and Medicine (SFRBM) 2013 Annual Meeting that relayed the findings from Baker Ruskinn’s extensive testing of HypoxyCOOL.