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Temperature, CO2, and pH in Cell Culture Media

What Seems Best for Incubation is Not Always Best for Handling

Most, but not all, cell culture media contain carbonate-based buffers which work with elevated gaseous carbon dioxide levels in the incubator to stabilize cell culture pH (see figure).  Carbonate-based buffers are present in vivo and so seem like an obvious choice for physiologically relevant incubation conditions. However, carbonate-based buffers can create non-optimal conditions for cultures during cell culture handling outside of the incubator. The critical cell parameters of temperature and carbon dioxide levels can affect carbonate buffered cell media. Continue reading to learn about the importance of having a cell culture growth medium during incubation.

The Basics – Is that orange red enough?

Proper pH is critical to cellular function. Aside from the proton-driven membrane transporters in cellular membranes and organelles, proper protein and lipid interactions are dependent upon available atomic and molecular charges.

Media are often formulated with phenol red as an easily-seen pH indicator. These solutions have a yellow color at or below pH 6.8. The color turns redder, then pinker as pH rises and a bright fuchsia above pH 8.2. Generally, an orange-red color indicates healthy pH conditions for mammalian cellular growth, pH 7.0 – 7.7. It is quickly obvious to the cell culturist that something is amiss with the culture if the color is bright yellow (too acidic) or fuchsia (too alkaline).

Cell culture pH can go outside of optimum ranges in static cultures in the incubator for several reasons. One reason is the build-up of acidic metabolic metabolites by cultures that have grown too dense or grown too long in that medium. Culture at low oxygen levels can also favor the production of lactic acid by cells, which will reduce culture pH. Contamination by fast-growing bacteria or fungi can quickly turn media yellow.

At the other end of the range, culture media that have been stored in the refrigerator for a month or more may increase in pH enough to turn fuchsia. If the CO2 tank feeding the incubator runs out, the cell medium also turns fuchsia.

pH Can Changes During Room Air Cell Handling Due to CO2 and Temperature Changes

Carbonate-based buffers in media are formulated for a specific CO2 gas level in the incubator. Users should consult the medium manufacturer for information and make sure that incubator settings and medium match.

Incubation pH is controlled by the high CO2 levels in the incubator (5% or more), but room air is far lower in CO2 (less than 1%). This leaves cells cultured in carbonic-based buffered media exposed to pH changes during cell handling in the room air BSC.

Water has a dissociation constant that decreases with decreasing temperature, unlike carbonate-based buffers. Temperature changes during cell handling in cooler room air means an additional component to pH swings.

Keep cells and media at incubation CO2 and constant physiologic CO2 and temperatures as much as possible for best pH control.

Additional Buffer Capacity for Cell Handling in Room Air

The addition of auxiliary buffers such as 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is a common approach to try to add more buffering capacity for cell handling in room air. HEPES has a dissociation constant that falls with falling temperature, similar to water. This makes it more compatible with a cooler environment outside the incubator and more able to maintain protein function in an aqueous medium than carbonate-based buffers [1]. However, HEPES buffer can generate toxic reactive oxygen species in combination with tryptophan and other medium components if the medium is not protected from light [2]. If you use HEPES buffer, keep stored medium bottles covered in aluminum foil to protect them from light. If possible, avoid use of HEPES altogether.

Work Fast

A standard room-temperature BSC is a hostile environment for cell cultures. Minimizing the time that cell cultures spend outside of optimum conditions is essential. Prepare all materials and equipment needed in advance of taking cell cultures out. Warm only the media that are needed to incubation temperature. Return the rest to cold storage.

Use a Gas and Temperature-Controlled Cell Handling Space

The use of a temperature-controlled and gas-controlled cell culture workspace, such as a hypoxia chamber or a barrier isolator, is by far the best way to keep cell cultures in their optimal environment during manipulations. With full-time optimization of conditions, cell cultures aren’t exposed to the pH swings cultures experience during cell handling in room air.

If you have any questions or feedback on cell culture media, please contact us here at the Cytocentric Blog.

1.         Baicu SC, Taylor MJ. Acid-base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency. Cryobiology. 2002;45(1):33-48.

2.         Zigler JS, Jr., Lepe-Zuniga JL, Vistica B, Gery I. Analysis of the cytotoxic effects of light-exposed HEPES-containing culture medium. In Vitro Cell Dev Biol. 1985;21(5):282-7.


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About The Author

Alicia D Henn, PhD, MBA

Alicia D Henn, PhD, MBA

Chief Scientific Officer of BioSpherix, Ltd



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