Why Your CO2 Incubator May Not Be Stabilizing | Blog

A reliable CO2 incubator is essential for consistent cell culture, but what happens when yours is not performing as expected? Maintaining precise environmental conditions, such as temperature, CO2 levels, and humidity, is crucial for preserving cell viability. Any fluctuations in these parameters can significantly impact your experimental outcomes, waste precious time, and undermine your results. Let's explore common reasons why CO2 incubators fail to stabilize and provide practical tips to get them back on track.

If your unit is not heating or cooling, it could be due to a broken element, controller, or a failed compressor for refrigerated incubators. These issues may require service, replacement parts, or a new machine altogether. Before shopping for a new unit, read on to explore other potential causes.

Key Parameters for Successful Cell Culture Growth.

Temperature:

For most cell cultures, the ideal temperature closely matches the human body’s natural temperature of 37°C. Maintaining cells within a precise range—just fractions of a degree above or below this threshold—is crucial to preserving their viability and ensuring consistent growth. Even slight deviations from this range can compromise cell health, slow down growth, alter cell metabolism and gene expression, and cause uncontrolled variability.

Minimizing door openings not only helps maintain a consistent temperature but also reduces the risk of contaminants entering the chamber. To further safeguard against accidental door openings, you might opt for an incubator with a built-in door lock.

As shown in the graph below, a 30-second door opening in an incubator can require over 30 minutes to fully recover. While a brief door opening may seem insignificant, even a few small interruptions can disrupt environmental stability, potentially compromising experimental reproducibility and cell viability.

Recovery graph of the IKS INB-203C incubator after a 30 seconds door opening. ¹

For additional details, you may visit Possible Reasons for Temperature Issues With Your Lab Incubator

Addressing Temperature Instability.

Potential cause 1: Temperature set point adjustments.

When multiple users operate the incubator, recent changes to the temperature set point may have been made without your knowledge, potentially requiring additional time for stabilization.

Potential solutions:

Verify the temperature set point and allow at least 2 hours for the incubator to stabilize.
Ensure the inner glass door latch is fully secured, as an improperly latched door can cause temperature fluctuations.

Potential cause 2: Prolonged inner door exposure.

Leaving the inner door open for an extended period allows external air to enter, disrupting the chamber's temperature.

Potential solutions:

Close the door and wait patiently for stabilization.
Minimize the amount of times the door is being opened as well as the total time the door is open. Keep a record of what is in your CO2 incubator and where, and know what you need to retrieve and where it is before opening the incubator.

Potential cause 3: Inner door gasket leakage.

Air leaks from the inner door gasket can cause temperature instability.

Potential solution:

Inspect the gasket to ensure it is securely in place with no gaps. Gaps at the joints can be sealed with silicon sealant, also check for deformation or tears in the gasket. If found, replace the gasket.

Importance of Temperature Calibration:

Over time, temperature sensors can lose accuracy and annual recalibration of temperature sensors is strongly recommended for consistent temperature readings.

How can you be sure your temperature sensor is always accurate? One reliable method is to use a secondary thermometer for verification.

If your incubator has a glass door, consider attaching a calibrated thermometer to the inside of the glass. This allows you to monitor the temperature without opening the door. By comparing the thermometer reading to the sensor's displayed temperature, you can quickly identify discrepancies, indicating if the sensor needs recalibration.

Temperature mapping is also another process for validating CO2 incubators to ensure uniform and consistent temperatures. It involves measuring and documenting temperature distribution to identify hot or cold spots, thus maintaining the proper environment for sensitive experiments like cell culture and microbiology. This process is vital for compliance with regulatory standards and to prevent variations that could compromise research results. Mapping can be performed after the CO2 incubator is calibrated.

For temperature mapping, it is important to allow the CO2 incubator to stabilize at the desired temperature before beginning data collection, which may take several hours depending on the CO2 incubator´s design and settings. Once stabilized, you can start recording temperature data regularly, typically every 15 minutes. Mapping duration might be 24 to 48 hours to capture any temperature fluctuations over time, and duration may last longer If more detailed analysis is needed.

Monitor and log CO₂ levels if necessary, as fluctuations in CO₂ concentration can influence temperature distribution. Once sufficient data has been collected, analyze the temperature readings to identify significant variations or patterns. Pay particular attention to any hot or cold spots and assess the overall temperature uniformity.

Evaluate the data in relation to the incubator’s specifications and the specific requirements of your experiments. Compare the recorded temperatures to the incubator’s set point and allowable tolerance range, ensuring all readings fall within acceptable limits. Investigate and resolve any deviations promptly.

Relative Humidity (RH):

Maintaining proper humidity levels in the growth chamber is vital to prevent medium desiccation. Although relative humidity (RH) levels can be as low as 75-80%, it is often necessary to keep RH above 90% to effectively support cell growth and preserve the culture environment.

When humidity levels in a CO2 incubator fail to stabilize, the evaporation of liquid growth medium can disrupt nutrient and waste concentrations, leading to inconsistent and unreliable results. Poor humidity control also contributes to condensate formation within the incubator chamber, creating an environment that promotes contamination. This not only compromises laboratory cleanliness but also hampers productivity, making effective humidification essential for reliable outcomes and a sterile workspace.

While some incubators offer a single, fixed humidity setting, others allow users to adjust and set precise humidity levels, which the system works to maintain.

However, real-world conditions often deviate from theoretical calculations, impacting the actual humidity levels delivered. Two key factors influence this discrepancy. First, since CO2 incubators are not fully sealed systems, ambient conditions play a significant role. Environmental factors such as temperature and ambient humidity differ between facilities. An incubator located in a cooler, drier laboratory is subject to greater challenges in maintaining a stable internal humidity level compared to one situated in a warmer, more humid setting. Additionally, routine actions, such as opening the incubator door disrupt internal environmental conditions, further compromising the system's ability to preserve consistent humidity.

Addressing humidity instability.

Potential cause 1: Watch your door openings:

When you replace the warm, humid air of an incubator with cooler room air, the cooler air contains less total moisture. As this air warms up, its relative humidity decreases because warmer air can hold more moisture.

Potential solutions:

Keeping the contents of the incubator organized and assigning shelves to a particular cell line or researcher can reduce the length of time the door remains open.
If the CO2 incubator will be used by various operatives, the inner glass door feature allows for easy viewing of inside samples and can help reducing time for door openings.

Inner glass door BIO Air Jacketed CO2 Incubator.

Potential cause 2: Check the water level in the humidity pan.

Potential solutions:

To quickly restore the %RH, try raising the height of your lowest shelf to improve air circulation around the water pan.
Ensure the pan is full, keeping the water level closer to the airflow for optimal humidity recovery.
Place a calibrated hygrometer inside the incubator to verify and compare its readings with the system's display.
Check the water level and refill the humidity pan with sterile distilled water once a week.

Potential cause 3: Check the filters.

Potential solution:

Make sure to change your filters regularly, based on your indoor air quality, to prevent clogs and maintain proper airflow.

Risks of Skipping Calibration:

Incorrect humidity levels.

Proper humidity levels are critical to prevent culture media from drying out and to maintain cell viability. On the other hand, high humidity levels can cause uncontrolled condensation, fostering the perfect conditions for contaminants to flourish.

Regular sensor calibration ensures accurate monitoring and control of humidity within the chamber.

Carbon Dioxide (CO2):

Cells thrive in environments with a specific pH range, typically between 7.0 and 7.7, to support optimal growth. To maintain this stability, growth media are supplemented with pH buffers, often utilizing a CO2-bicarbonate system. Atmospheric CO2 interacts with humidity to form carbonic acid, which can influence the pH of the growth medium. By carefully regulating atmospheric CO2 levels, the pH of the growth medium can be kept stable, ensuring a suitable environment for cellular growth.

Atmospheric CO2 interacts with humidity to form carbonic acid, which can influence the pH of the growth medium. ²

A commonly referenced value is 5 percent, compared to the typical 0.3 percent concentration of CO2 present in the atmosphere at sea level. The precise percentage required will depend on the specific type of growth medium. Insufficient CO2 in the atmosphere may result in the gas escaping from the growth medium, causing the mixture to become excessively alkaline. Conversely, an excess of CO2 in the atmosphere may lead to greater absorption by the medium, ultimately rendering it too acidic. Regardless of the required concentration, the incubator must be capable of accurately calculating the atmospheric CO2 levels.

CO2 incubator not stabilizing CO2:

Potential cause 1: Defective CO2 sensor:

A malfunctioning sensor may provide inaccurate CO2 readings, leading to unstable levels within the incubator.

Infrared (IR) CO2 sensors work by detecting how gases absorb light at specific frequencies. Higher CO2 levels block more IR rays, reducing what the sensor detects, while lower levels let more rays through. These sensors provide accurate, reliable, and stable measurements even in changing environmental conditions.

Thermal conductivity (TC) sensors measure CO2 by comparing its resistance to ambient air, which acts as a reference. They detect resistance changes caused by CO2 flow but are highly sensitive to humidity and temperature, which affect air resistance. Actions like opening a CO2 incubator door can significantly reduce the TC sensor's accuracy.

Both sensors can measure CO2 in incubators, but their performance varies. Thermal conductivity (TC) sensors are an affordable option, but their measurements can be impacted by fluctuations in temperature, humidity, and gas composition. In contrast, infrared (IR) sensors provide superior accuracy, remaining unaffected by external environmental factors.

Potential solutions:

Use a CO₂ gas analyzer to confirm the concentration levels. If needed, calibrate the incubator’s gas injection system to achieve the required settings.
Gas supply of the CO2 supply system should be checked on a daily basis to ensure the operativeness and the operational safety of the incubator.

SCO Air Jacket CO2 Incubators SCO5A Standard with IR CO2 sensor.

Potential cause 2: Blocked gas supply as of regulator or cylinder valve issues.

Possible causes for a blocked gas supply, might be:

Partially opened valve: If the CO₂ cylinder valve is not fully opened, gas flow can be severely limited.
Faulty or clogged regulator: Debris or moisture in the regulator can restrict flow. Single-stage regulators are also more prone to pressure fluctuations.
Regulator freeze-up: Rapid gas expansion can cause ice to form inside or around the regulator, especially if the cylinder is nearly empty or the lab is cold.

Potential solutions:

Inspect the gas supply system and CO2 input pressure, including the cylinder, connections, and hose system. Any blockages or low pressure can disrupt the flow of CO2.
Monitor the CO₂/N₂ gas tank level.
Review the gas pressure hose and make sure it is connected correctly to the sleeve of the gas supply system, ensuring it is secured by a hose clamp.

Potential cause 3: Gas supply pressure.

Gas supply pressure issues might occur for different reasons:

Low gas supply: Inspect the pressure gauge on the two-stage regulator daily to confirm it stays close to 15 psig. Possible causes for low gas supply might be: pre-pressure low, supply line blocked or No CO2.

Potential solutions:

Connect a new gas cylinder if pressure drops below 15 psg or 1 bar.

Raise pre-pressure to 1 bar.
Check supply line to device.

Overpressure: The operating pressure of the gas applied to the CO2 incubator must not exceed 1 bar. If the gas is supplied at a higher pressure, the valves may not close correctly and the gas supply control may be impaired.

Potential solution:

Set the gas supply to a range between 0.8 bar min. and 1.0 bar max. and make sure that this pressure setting cannot be changed.

Gas regulators for SureTherm™ CO₂ Incubators.

Gas pressure hose installation.³

Risks of Skipping Calibration:

Inaccurate CO2 and O2 levels.

CO2 and O2 levels are vital for replicating physiological conditions within the incubator. Like temperature sensors, the performance of gas sensors degrades over time. To ensure accuracy, CO2 sensors should be recalibrated annually. O2 sensors should be recalibrated every six months. Not all CO2 incubators come equipped with an O2 sensor. Standard models typically include sensors for CO2, temperature, and humidity. In contrast, tri-gas incubators are designed with O2 sensors and are commonly used for specialized applications such as embryo culture.

Proper CO2 Incubator Use for Improved Stability and Performance.

Ensure there is sufficient water in the pan by checking weekly. Check the water level and refill the humidity pan with sterile distilled water once a week or as required.
Provide adequate spacing between samples when loading the incubator and avoid placing items against the incubator’s side walls, as this can prolong the time required to reach the desired temperature.
Follow the required room temperature, humidity and environmental conditions indicated in the user manual to maximize the use of the CO₂ incubator to its best performance.
Gas supply of the CO2 supply system should be checked daily.
Maintain a well-organized interior to quickly locate cell cultures, reducing the need for prolonged or frequent door openings. This simple step helps prevent airborne microorganisms from entering the chamber. Depending on your lab's workflow, there are various effective ways to organize the incubator's contents to suit your needs.

(A) Dedicated incubators for keeping cell lines and primary cells separate.

(B) Dedicated shelves for different cell lines.

The tips on this article are intended as general guidelines and should not substitute for consulting your CO2 incubator's user manual or seeking technical support from the manufacturer.

References:

https://www.xiltrixusa.com/resources/co2-incubator-monitoring
https://www.nuaire.com/resources/direct-heat-or-water-jacketed-co2-incubators-white-paper
https://www.marshallscientific.com/v/vspfiles/specs/VWR%20Air%20Jacketed%20CO2%20Incubator%20with%20Sterilization%20Cycle%2C%20Cat.%20%23%2010810-888%20Manual%20-%20Marshall%20Scientific.pdf
https://www.eppendorf.com/product-media/doc/en/604516/CO2-Incubators_White-Paper_051_CellXpert-CO2-Incubators_CO2-Incubator-Maintenance-Guide-Set-up-Care.pdf