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In the previous articles, we discussed about the significant role of amplitude, frequency, and temperature control of incubator shakers in cell culture. By scientifically adjust these parameters, researchers are able to optimize cell growth conditions and improve the reliability and reproducibility of experiments. This article continues to discuss how humidity control and CO₂ concentration control in incubator shakers affect cell culture. These two parameters are also critical in the cell culture environment, as they are directly related to the stability of the medium and the health of the cells, which in turn affects the accuracy of the experimental results.

Humidity control

The effect of humidity in incubator shaker on cell culture is mainly reflected in its effect on cell growth and metabolism.

We all know that the common methods of cell culture are adherent cell culture and suspension cell culture. Although the culture methods are different, but there are very high requirements for humidity, and the general cell culture needs to have a high humidity, so as to avoid the dehydration of the medium in the process of cell culture, which affects the culture effect and even leads to the failure of cell culture.

For example, experiments in the culture of rat fibroblasts under different relative humidity (RH) conditions have shown that humidity has a significant effect on cell growth. Within 48 hours after inoculation, suitable humidity conditions can significantly promote cell growth and metabolism, while poor humidity may lead to stunted cell growth and even death.

In incubator shakers, preventing media evaporation becomes key to humidity control because the gas flows more violently than in the incubator. Although saturated humidity is effective in preventing evaporation, it is also prone to condensation in slightly lower temperature regions. Excessive, persistent condensation can harbor bacteria, contaminate the environment, and even clog the breathable membrane at the mouth of the culture bottle, further hindering cell growth and metabolism.

For this purpose, in incubator shaker, the ideal state for humidity control is to reach a humidity close to saturation but avoid condensation.

The specific strategies are as follows:

●When using large-volume culture medium, the humidity is controlled between 70%~80% to inhibit the formation of condensate;

●When using a small amount of culture medium (such as test tubes or culture plates), the humidity is controlled between 80%~90% to prevent evaporation, even if a small amount of condensation is generated, it is still controllable.

However, the normal passive humidification of the sink can lead to fluctuations in the humidity value, mainly depending on the tightness of the cavity and the number of containers: it is difficult to maintain the humidity when the cavity is poorly sealed or there are few containers; When the seal is good or there are many containers, the humidity may be too high and condensation may occur. To avoid these adverse situations, it is recommended to employ an active humidification or humidity control system to further precisely regulate the humidity and ensure the stability of the cell culture environment.

The commonly used temperature in animal cell culture is 37 °C, but there are some cell suspension culture temperatures lower than 37 °C, such as insect cells/fish cell culture, etc., when the temperature is controlled by incubator shaker, sometimes the compressor will start to cool down, which will form a strong dehumidification effect, and the passive humidification of the sink will produce less water vapor due to evaporation in the low temperature environment, and the humidity in incubator shaker will also become smaller, so it is difficult to place the water tank to meet the requirements of the humidity required for cell culture, which affects the culture effect of cells. The method of active humidification or active humidity control can achieve the effect of accurate humidity control in incubator shaker.

In summary, humidity control of incubator shaker is critical to the success of cell culture, and humidity levels directly affect cell growth, metabolism, and the accuracy of experimental results. Therefore, during the cell culture process, the humidity in the shaker needs to be closely monitored and precisely adjusted to ensure that the cells grow under optimal conditions, so as to improve the reliability and success of the experiment.

Effect of CO2 concentration control on cell culture in incubator shaker

CO2 concentration control in incubator shaker plays an important role in cell culture, which is mainly reflected in the following aspects:

● Maintain normal cell metabolism:

CO₂ is one of the products of cellular metabolism. CO₂ produced by cellular metabolism is transported through the bloodstream to the lungs and excreted from the body, and in a cell culture environment, maintaining proper CO₂ concentrations is essential for the normal metabolism and growth of many cells. In the absence of a proper CO₂ environment, cell metabolism may be blocked, which in turn can lead to poor cell growth and even death.

●Adjust pH value:

CO₂ is essential for the pH regulation of the medium. In a CO2 incubator, the appropriate CO₂ concentration keeps the pH of the medium stable, providing an ideal growth environment for the cells and ensuring that their metabolic activities run smoothly.

●Affects cell growth and metabolism :

The CO₂ concentration directly affects the growth and metabolism of cells, and high or low CO₂ levels can adversely affect cell growth. By precisely controlling the CO₂ concentration, the cells can be prepared to grow in a suitable environment and ensure healthy growth in incubator  shaker.

In summary, the carbon dioxide environment in incubator shaker plays a crucial role in cell culture, which provides a in vivo-like growth environment for cells by maintaining appropriate carbon dioxide concentrations and adjusting pH values, thereby supporting the normal growth and metabolism of cells.

Understanding the effects of humidity control and CO₂ concentration control on cell culture can help users better optimize and apply incubator shakers to provide a more suitable environment for cell growth. By scientifically and effectively adjusting these parameters, users can effectively prevent adverse factors such as medium evaporation and condensate accumulation, and stabilize the pH value of the medium to ensure the normal metabolism of cells. This knowledge not only improves the success rate of experiments, but also greatly improves the accuracy and reliability of culture results, laying a solid foundation for subsequent experiments.