The ability to have a definable path to a procedure creates fewer variables to consider when analyzing results. A CO2incubator is one tool scientists rely upon in order to provide a controlled environment for cell growth.
Conditions within a CO2 incubator, however, may vary from one supplier to another, or even from one incubator to another. The environment under which cells are grown may not always be optimally controlled. This can raise more questions related to the integrity of the results and ultimately the answers to the questions that scientists seek.
As such, CO2 incubators are a crucial component to the method employed within most scientific experiments and are a risk which should be mitigated, or better understood, in order to enhance the reliability and repeatability of the experiment.
Temperature control is a particularly important environmental parameter for optimal cell growth, because growth or a reaction in a culture may be influenced by subtle changes (or varying fluctuations) in temperature throughout the interior chamber. Whether a protein will be expressed or not expressed can be contingent upon a difference of just a few degrees. The inability to provide precise and stable control over temperature may result in different or abnormal growth and production results (e.g., transfection rates in gene insertion experiments can be affected by variations in temperature; so too can the yield of these insertions when protein expression from a bacterial colony expressing a transfected fragment is producing at a lower rate). This can lead researchers down the wrong path and/or impact the repeatability or reliability of the results.
Additionally, the environmental parameters controlled within a CO2 incubator (CO2, RH, and temperature) define the conditions for cell growth, but control technologies do not always account for the fact that each parameter will affect the other. For instance, temperature and a system’s ability to provide temperature control influences the relative humidity (RH) and CO2 levels that also impact the condition an experiment requires. With respect to RH, if the temperature inside the incubator is not properly maintained, the amount of water suspended in vapor will vary. And, as temperature fluctuates – especially as it cools – moisture will accumulate on surfaces with a temperature below dew point. This condensation is a hallmark of poor environmental control, an undefined variable that will make any experiment less reliable and repeatable. Not to mention that it will invite further unknowns or potential contaminants in the system.
Fluctuations in temperature will also affect CO2 concentration. CO2 is an acidic molecule. Increases in CO2 within an incubator will lead to a less neutral environment that will, in turn, impact the cultures exposed to it. If the temperature within an incubator cannot be precisely controlled or maintained, the increase in CO2 content can be detrimental. As temperature increases, so does the amount of CO2 in the expanded air and the level of acidity that cultures can be exposed to. Further, CO2 is soluble in water. This will provide acidic water vapor to be circulated around the chamber. Variations in temperature will impact the rate of solubility of CO2 within the chamber. As temperature is ramped up and brought back down, the CO2 molecules are absorbed in the moist environment and shed through condensation into the water source and onto surfaces as the air mass inside the incubator is cooled. As such, the increase of the solubilized CO2 and the acidified water it creates can lead to a hostile environment that can destroy a tissue culture or other experiment that depends on a controlled environment (e.g., 37°C, 5% CO2, 95% RH) and consistent pH of 7.4 for growth.
The concept of providing a controlled environment for cell growth is not new. The level of control, or defining the acceptable level of control, is always the challenge for any experiment. Many researchers are keenly aware that the CO2 incubators used in their labs all have variations in temperature throughout the chamber, from shelf to shelf and front to back. To combat this, some suppliers provide operating instructions indicating that the only way to maintain their specified environmental conditions and temperature control (stability, uniformity, accuracy) is to open the incubator door several times a day. Coincidentally, that same supplier explains that doing so will help reduce the accumulation of water on the interior surfaces of the incubator. But, in addition to the risk of overruns in temperature (which in turn affects humidity and CO2), this same instruction will increase the risk of introducing another threat to the integrity of the experiment – contamination.
Also, laboratories must often develop their own procedures to control the effects of inadequate CO2 incubator function, often adopting protocols and work-arounds to fit the lab that it is used in. If the temperature in the CO2 incubator is not consistent, and if the system is not capable of maintaining the environment through calibrated and accurate controls, then the ability to recover from lab use is a daunting task. For example, during heavy use the instrument must compensate for door openings and closings, which can cause an overshoot of heating elements trying to recover temperature quickly without regard to rapid temperature changes. This aggressive approach to temperature recovery can expose cultures to conditions that can alter a result or pathway. Even control of temperature during recovery periods will provide a more stable, predictable condition. This is understood, but finding the right instrument is difficult. Limiting access can maintain the conditions, but that is not realistic in most labs.
Poor temperature uniformity within a CO2 incubator chamber is another downfall in technology experienced within some systems. The number of heating elements an incubator utilizes, as well as their placement within the interior chamber, can lead to variability within certain areas of the incubator, resulting in cold spots and heated spots throughout the system. Many researchers have learned to accept this limitation and have adapted to this by relying on a favorite spot (commonly referred to as “sweet spots”) within the chamber where they feel more consistent or reliable conditions are delivered. This is a common characteristic found within many CO2 incubators. This, however, makes it impossible to effectively use the entire space of the incubator, sacrificing he usable workspace available to researchers and, ultimately, the productivity of their laboratory.This should certainly be evaluated prior to the purchase of a new CO2 incubator. The work space within a controlled environment should not be a patchwork of conditions that each laboratory has to analyze to reasonably ensure reliable and repeatable results. More uniform temperature allows maximum use of available space, as well as a better-humidified environment that will bring more accurate CO2levels and appropriate pH. Moreover, this will also assist in better control of condensation and water collection on plates and flasks, since the temperature is not constantly fluctuating.
To remedy these stability and uniformity problems, look for control algorithms that can predict times of heavy use by accounting for the habits of the user and compensating for those patterns accordingly. Look for a closed feedback loop controller that is programmed to adapt to the users in the laboratory and their practices of opening and closing the incubator, not an assumption of use without a real basis for comparison that assumes all laboratories have the same user patterns. Additionally, look for unidirectional airflow which helps provide a consistent temperature throughout the chamber. Finally, a sufficient number of heating elements and even placement within the interior chamber will help maintain stability and reduce temperature variation from shelf to shelf and front to back of the chamber.
A few suppliers have developed control algorithms designed to address these concerns and to better control the environment within an incubator. Some, however, only take into consideration the control of two variables – temperature and CO2. This approach does not consider the impact of, or a lack of control over, the other variable: relative humidity. This is critical to providing an optimal environment for cell growth and contamination control. As temperature increases within the chamber, the expansion of the air will allow more water vapor to be held and the RH will increase due to this capacity changing the equation. Any drop in temperature will cause this excess moisture that had been held to drop out of the air space and condense on a cooler surface (e.g., culture dishes, assay plates, or the shelves).
The environment within an incubator is complex with all factors affecting each other in subtle and overt ways. The inability to accurately control that environment results in a heightened potential for contamination, unreliable and unrepeatable results, inconsistent environmental parameters, and the incorrect belief that certain conditions are always met and can used to define an experiment. Creating an algorithm that adjusts and predicts usage of the heating elements present creates a more stable and uniform temperature range, which flows into a stable and controlled humidity and an environment with an appropriate pH. Scientists should evaluate the way in which temperature – and each variable critical to cell growth – is controlled within their current incubator and any new incubator they are considering.
Baker’s proprietary CO2 incubator control algorithm, InteliCELL™, helps to create superior temperature uniformity and stability inside Cultivo™.