Understanding Cell Culture Humidity: Practical Tips For Maintaining Healthy Cell Cultures
Maintaining healthy cell cultures can be a difficult balancing act, with cell integrity and proliferation depending on the careful fine-tuning of multiple parameters, including temperature, gas composition, and humidity.
Among these three parameters, temperature and gas composition often take center stage, and researchers can overlook cell culture humidity. But without adequate humidity levels, cultures can quickly become compromised, ultimately impacting the reliability and reproducibility of experimental results.
That said, maintaining proper humidity levels isn’t always straightforward and can present numerous challenges. Here, we explore in more detail why humidity control matters and offer some practical steps you can take to ensure your cultures stay protected.
Why Cell Culture Humidity Matters
Preventing the Evaporation of Culture Media
Cell cultures rely on consistent concentrations of salts, minerals, and essential amino acids in their culture media to support normal growth and functioning. If the cell culture humidity dips below 85–95%, the culture media can evaporate, increasing the concentration of the media composition and disrupting the delicate balance of the proper culture environment.
Changes to media composition can alter cell growth in various ways, including increases or decreases in proliferation and up- or downregulation of gene expression.1 These inconsistencies can cause variation in study results, which can impact study reproducibility. In extreme cases, media evaporation can lead to cell death, prolonging study timelines and driving up costs.
A well-documented consequence of cell culture media evaporation is known as the "edge effect," whereby, in multiwell plates, the wells at the outer edge experience higher evaporation rates than the inner wells. This uneven evaporation makes the edge wells more prone to variability.2 Additionally, edge wells are also more sensitive to temperature changes, cooling and warming more quickly when plates are moved in and out of the incubator2. These factors can result in variations in cell density, proliferation, and gene expression across the plate, potentially undermining reproducibility and increasing repeated experiments.
Reducing Contamination Risk
Excessively high cell culture humidity can lead to a build-up of condensation on the interior surfaces of the incubator or the underside of culture vessel lids, creating the optimal environmental conditions for microbial growth.
These microbes can contaminate cell cultures, leading to changes in cell behavior, morphology, and potential cell death, impacting the reliability of results.
Key Considerations for Maintaining Consistent Cell Culture Humidity
Reduce Disruption from Door Openings
Humidity in a cell culture incubator can be disrupted by something as simple and routine as opening the incubator door. Each time the door is opened, typically drier, ambient room air replaces humidified air inside the incubator. The longer the door is open, the greater the change to the internal environment.
The extent of humidity change depends not only on how long the door remains open, but also on how quickly the door is opened and closed. A faster opening leads to more airflow disruption and a sharper drop in humidity.
Tips for minimizing disruption from door openings:
Before purchasing an incubator:
- Evaluate recovery performance: check how quickly an incubator can restore humidity after door openings. Be aware of variations in testing conditions that may affect results. For expert guidance, see the Decoding the Data eBook.
For existing incubators in your lab:
- Minimize door openings: a simple way to help maintain incubator humidity is to only access the incubator when absolutely needed, and avoid leaving the door open for prolonged periods
- Open the door slowly: a slower, more controlled opening reduces the volume of dry air entering the chamber, helping to shorten humidity recovery time
Use the Right Water Type
Not all water is suitable for use in an incubator. While high-purity options like deionized or Type 1 water may seem ideal for avoiding contaminants, their low ionic content can corrode internal components such as stainless steel. Over time, this corrosion can degrade incubator performance, impacting its ability to maintain the appropriate humidity levels.
To help ensure your incubator remains in optimal condition for maintaining humidity, you should only use sterile, distilled water with a pH between 7 and 9.
Properly Maintain Water Reservoirs
Even with the right water, issues can arise. Low water levels or stagnant water can hinder humidity control and encourage microbial growth.
To make sure water levels are optimally maintained, be sure to regularly check and refill the water reservoir as needed. Beyond just simply topping up water levels, you should also change the water every 1-2 weeks to reduce the risk of microbe growth.
Maintain Stable Incubator Temperature
Incubator temperature and humidity are closely linked, with even minor temperature fluctuations causing shifts in humidity. For instance, rises in temperature can increase evaporation of culture media, whereas drops in temperature can cause condensation to develop.
These temperature-driven shifts may override the strategies put in place to maintain optimal humidity, making temperature maintenance essential.
Tips to help maintain stable temperature:
Before purchasing an incubator:
- Look for features that aid temperature uniformity: incubators with direct heat systems distribute heat evenly around the chamber, helping to prevent cold spots and reducing the likelihood of condensation. Additionally, outer-door heaters that warm the glass door relative to ambient temperature can further promote uniformity and minimize the chance of surface condensation
- Consider air jacketed over water jacketed incubators: air jacketed incubator models offer faster temperature recovery after door openings than water jacketed models due their ability to adjust temperature on/off cycles based on the air temperature inside the chamber. You can find more information on air jacketed vs water jacketed incubators here.
- Consider humidity mitigation features: some incubators incorporate dew sticks or Peltier elements that direct condensation to controlled areas within the incubator, preventing it from forming unpredictably on surfaces or vessel lids. In certain models, this collected condensation can be sterilized and reused for humidification, particularly in systems with UV germicidal lamps that decontaminate the water in the humidifying pan.
For existing incubators:
- Be mindful of external factors that could influence temperature: significant temperature fluctuations in the incubator's external environment could impact internal temperature. Avoid placing incubators near HVAC vents, windows, or heat-generating equipment, and try to minimize large temperature changes in the room (e.g., through air conditioning cycles)
- Reduce the frequency and length of door openings: as previously mentioned, door openings can disrupt the internal incubator environment, in terms of both humidity and temperature, so avoid frequent and prolonged door openings
Cell Culture Humidity Control as a Foundation for Culture Success
Maintaining consistent incubator humidity is essential for healthy cell cultures and reproducible results.
Fortunately, with thoughtful equipment selection and disciplined lab practices, researchers can control humidity effectively and protect their experimental outcomes.
Looking for more effective humidity control in an incubator? Explore how incubator design can make a difference PHCbi incubators are built to support optimal humidity control and easier maintenance for consistent cell culture conditions.
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