The Significance of pH Stability for Cell Cultures

Cell culture media generally comprise amino acids, salts, vitamins, other substances, and an anorganic buffer substance (NaHCO3). To allow cell cultures to grow, the pH value should be balanced and stable. Starting values of pH 7.2–7.4 are ideal for nearly all cells; these values can vary from cell type to cell type during cultivation. The pH value of NaHCO3 buffered media depends strongly on the CO2 content of the incubator atmosphere. Depending on the selected medium, it is necessary to add a corresponding amount of CO2 concentration. A continuous supply of CO2 is required for pH stability.

How a CO2 incubator guarantees pH stability

Figure 1 - BINDER CB CO2 incubator.

Figure 2 - Rapid stabilization of the pH value after door opening in a BINDER CB chamber with an IR sensor versus long recovery times in models with thermal conductivity sensor technology.

For successful cell cultivation, it is absolutely necessary that the pH stability be maintained throughout the entire culturing process. For example, even a slight disturbance, such as the opening of an incubator door, can cause a disturbance. This is a common occurrence, particularly if several persons are using the same incubator. Even if the door is opened for only 30 sec, the parameters temperature, humidity, and CO2 concentration in the chamber interior are affected. The CO2 concentration immediately drops to about 0.3% (normal atmosphere), which in itself causes a pH value shift within the growth medium. If the system does not counteract this immediately, it may have negative consequences for cell growth.

Only a highly dynamic and adaptive system with an IR sensor, such as the BINDER CB (Binder GmbH, Tuttlingen, Germany) (Figure 1), can rapidly compensate for such disturbances in the climate balance—generally within a few minutes. The advantage is that the measurement is independent of the air moisture content and can be made correctly at any time, and the CO2 content can be immediately regulated. The buildup of humidity in the air is usually relatively slow after disturbances such as a door opening, due to the physical conditions. However, as a result of the rapid recovery times in a BINDER CB with IR sensor technology, the pH value remains within the neutral range, while long recovery times in units with thermal conductivity sensors can lead to cell-damaging values (see Figure 2). This effect is further increased by frequent door openings.

Incubator protection

Figure 3 - Possible consequences of a disturbance in the CO2 supply on the pH value of a cell culture: The pH value could shift into the alkaline range with insufficient gas supply (low CO2 level), or into the acid range with excessive gas supply (high CO2 level). Intelligent Fail Safe ensures rapid stabilization.

The above example demonstrates how cell cultures react to a disturbance in CO2 concentration. Ensuring a constantly regulated CO2 supply is critical to the safety of the cells in an incubator. At any time during the long cultivation period, it is important to prevent shifts in the pH value into the acid or alkaline range beyond the tolerance limits (see Figure 3). After a shift of only 4 hr, negative effects on growth can be expected. The consequence is slowed cell division, which can eventually endanger projects and even lead to complete cell death.

Figure 4 - Rapid adaptation of the CO2 supply due to activation of Intelligent Fail Safe.

It is not often that the CO2 content in an incubator changes unnoticed, but it happens more often than one would think. There can be numerous reasons for this. A problem that occurs over the weekend, when, for example, there is no one in the laboratory, may prove irreversible. A good solution is the Intelligent Fail Safe system (Binder). During normal incubator operation, it stores information on gas consumption, which is normally necessary to obtain the selected CO2 concentration. During continued operation of the unit, the actual gas supply is then constantly compared to the saved value. If there are deviations in the CO2 injection, i.e., too much or too little gas, Intelligent Fail Safe takes over control of the CO2 supply and returns the value to the original concentration (see Figure 4). The system reacts immediately to any change in the gas supply, and it does so before an alarm for CO2 over- or underconcentration is triggered (±1 vol % of the target value). The pH value is rapidly stabilized again. The stability of the pH value is therefore guaranteed in every case, even during a controller or sensor defect. The Fail Safe system is referred to as “intelligent” because it independently and continuously monitors the CO2 concentration.

Cell culture safety

The Intelligent Fail Safe system for monitoring CO2 concentration in a Binder CO2 incubator works in a similar way to overtemperature protection systems incorporated in any general laboratory instrument today. It is an effective safety function for pH stability in CO2 incubators that protects valuable work or samples. The system is suitable for cell culture specialists who cannot afford to leave anything to chance in their applications.

Mrs. Maurer is Product Manager, Binder, Bergstr. 14, D-78532, Tuttlingen, Germany; tel.: +49 7462 2005 0; fax: +49 7462 2005 100; e-mail: info@binder-world.com; home page: www.binder-world.com.

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