If you live long enough, you get to see several things go from being labeled as good then to bad and often back again. I’ve seen it with coffee and eggs, which have alternately been seen neutrally, been lauded or been demonized. Many folks are long-lived enough to remember when cigarettes were advertised as being good for you, complete with physician endorsements.
And then there’s oxygen. Can’t live with it; can’t live without it. We need a certain amount of it in the air so that our lungs can send it to our bloodstream, and yet if we breathe too much pure oxygen (or even just high levels of it) for too long, we won’t be shuffling around in our mortal coils much longer. And the reason antioxidant products are often popular is because free oxygen radicals in our bodies can cause cellular damage over time.
So it is, then, that oxygen also is important in life-sciences research and pharma/biotech R&D, though perhaps it's been seen as an enemy at times or not given the attenion it deserves at others. It’s the carbon dioxide (CO2), for example, that people traditionally think more about in terms of things like cell culturing, but according to Carl Radosevich, business intelligence and product manager for Panasonic Healthcare Corporation of North America, more attention needs to be paid to oxygen levels as well when working with cells. And so we talked a bit on the topic.
DDNews: To start, while this may be rudimentary for many readers, explain why, as cell culture techniques are refined there is such a need to simulate accurate in-vivo conditions within an in-vitro environment.
Carl Radosevich: I think this is always a great question to reflect upon, as culturing cells in vitro becomes more and more important in the research and clinical fields. Cell culture techniques are often utilized to accumulate the primary data set for research scientists to study the genetic, molecular and cellular mechanisms of a given tissue or system. The data gathered from cell culture, whether a phenotypic cellular observation or a gene expression analysis, acts as a compass for future investigations. To have this “compass” point in the most accurate direction, in-vitro cells must be populated in most human-like conditions. It’s a simple concept that becomes relatively complex in practice.
DDNews: Before we get to the issue of oxygenation specifically, explain some of the key improvements in cell culture techniques over the years, both in terms of the long term and also, in much more recent years, what leaps forward have we seen?
Radosevich: In terms of cell proliferation techniques in a static culturing system, there has been a wealth of culturing tools that have allowed for significant advancements in vitro. Engineering three-dimensional platforms for cells in vitro allows for a lot more insight with how cells react within an extra-cellular matrix or how, for example, cancerous cells metastasize within a tissue. 3D techniques have added an important layer of simulating the in-vivo anatomy and will become increasingly significant in years to come. Of course, we’re also seeing some great improvements within media optimization that allow for a more pinpointed formulation from cell line to cell line. I believe a great challenge lies ahead, in both static and dynamic cell culture, for elucidating the specific nutritional requirements for individual cell lines and manufacturing effective media, free of animal-derived components.
As for CO2 incubator technology, we have come a long way in tightly controlling key parameters that regulate culture conditions. As mammalian-based cell culture research has migrated towards more sensitive applications, the need for a precisely controlled environment has skyrocketed. One aspect of mimicking in-vivo conditions within an incubator requires a uniform distribution of carbon dioxide and oxygen gases, relative humidity and temperature. This is achieved through sophisticated gas sensor, airflow, insulation and heater systems, all controlled by “smart” microprocessor systems that react, in real time, if these conditions are altered.
DDNews: Again, before delving too much into oxygen specifically, what is the relative importance of previous advances and more recent ones in oxygenation to cell culturing in general, and also to oncology research more particularly?
Radosevich: This goes back to the idea of producing the best in-vivo environments in vitro. The importance of these advancements at all levels of cell culture allow for a more precise and effective continuum of elucidation of cellular events to drug targeting and discovery to therapeutic development, clinical trials and so on. The truer the dataset that we can achieve upstream in the lengthy therapeutic development timeline, the greater likelihood we’ll have with developing successful therapeutics at the end. This will also streamline processes and save costs.
Scientists must demand more and more precise technologies for culturing cells. This includes the requirement for precision technology within the chamber that your cultures spend the most time: your CO2/O2 incubator.
DDNews: Now, to oxygenation more directly. How does a better understanding of how oxygen factors into biological mechanisms advance oncology research development with regard to cell cultures?
Radosevich: Understanding the role oxygen plays in your biological system may lead to a more holistic blueprint of sub-cellular interactions within your tissue of interest. Countless studies have demonstrated that the oxygen level controls specific biological events, such as cell differentiation, cell proliferation and apoptosis. Oxygen plays more of a role than just a “damaging agent” but rather acts as a molecular signal for a variety of key cellular events. For example, the HIF protein, which is expressed at hypoxic conditions, regulates the transcription of a variety of genes critical for maintaining overall cellular homeostasis and key pathways. Since many tumors progress at hypoxic cellular conditions, understanding the role of oxygen levels within the cancer biology is paramount to research investigations.
DDNews: What are some of the critical issues that researchers need to understand with oxygen levels?
Radosevich: In oncology research, understanding specific levels of oxygen at which tumors develop or metastasize is critical, since these events are often hypoxic in nature. Furthermore, pathological tissue like cancer contains an oxygen gradient in which inter-cellular oxygen levels can reach below 1 percent. Developing cell culture platforms for oncology research requires a detailed examination at these oxygen levels; proper research equipment should be in place to facilitate such culture conditions.
DDNews: What are some of the biggest hurdles to overcome in maintaining cell homeostasis with regard to oxygen levels and what are the best ways to overcome these hurdles?
Radosevich: For some researchers, a big hurdle is simply adopting a new methodology to incorporate oxygen control within their experimental systems. Historically, cell lines were not conventionally cultured under hypoxic conditions and seemed to be viable model systems. However, as the literature has shown us, hyperoxic-exposed cells may provide erroneous data at many biological levels. This is why key areas of life science, like regenerative medicine and cancer research, are leading the charge with the utilization of hypoxia methods. Additionally, a big hurdle with oxygen in the past was the lack of reliable instrumentation and resources for this type of control. New, cutting-edge incubation equipment that tightly regulates oxygen and other key conditions are now available to researchers in a variety of options.
DDNews: How does all this factor into making decisions about which cell culture incubator equipment to choose?
Radosevich: The key is to look for culture incubators with excellent experimental versatility. Day-to-day culturing methods are not uniform in nature, so it may be worthwhile to use an instrument that adapts to you needs. For example, if your lab uses both hardy, “workhorse” cell lines that have been transformed enough to withstand ambient conditions and primary cells, a CO2/O2 incubator allows for an easy conversion between differently gassed states to facilitate the needs of each system. Equally important, look for an incubator that has been designed for repeatability. To get consistent cell growth and experimental replication, your incubator must be smart and powerful enough to detect environmental deviations (i.e. door openings) and recover the in-vivo-like environment quickly. This will help eliminate discrepancies in your data and allow for better consistencies in with your cell growth and experimental repeatability.
DDNews: Do you see O2 demands supplanting CO2 demands in terms of industry norms for cell culture incubators, or is this more of an equal, hand-in-hand issue where both need to be merged into one industry standard?
Radosevich: For researchers working to elucidate the mechanisms of previously unknown biological systems, I believe oxygen control will equate to carbon dioxide control for the best maintenance of a homeostatic environment. In the biopharmaceutical world, where established cell lines are primarily used as factories for biologic production, oxygen control may not be a priority. Much like your culture media requires a tailored formulation for optimal cell growth and viability, the conditions within your cell culture incubator should be assessed and tailored for each system’s needs.
DDNews: Is there anything else you’d like to add in closing on this subject?
Radosevich: Regardless of your application or system of study, evaluate the role that oxygen may play with your cell health. At Panasonic, we have been designing and manufacturing cell culture equipment for over 30 years. For the majority of that time, we have provided oxygen-controlled units to high demand markets such as IVF clinics and embryology researchers. With the revolution of the stem cell community and ensuing clinical and translational applications, Panasonic began working closely with world stem cell leaders, including the Dr. Yamanaka from Kyoto University in Japan and Dr. Herzenberg’s Lab at Stanford University. Both teams confirmed the necessity for doing research in the PhysOx environment when culturing stem cells. Many other areas of stem cell science are following suit with hypoxia studies, demonstrating key benefits when testing under these conditions.