The Essential Guide to HEPES in Cell Culture

Buffering refers to the ability of a solution to resist changes in pH. Buffers typically consist of a weak acid and its conjugate base, or a weak base and its conjugate acid, which work together to neutralize added acids or bases. To mitigate the acidity generated by high CO₂ levels in incubators, chemical buffers are added to cell culture media. Maintaining a stable pH is crucial for optimal cell function and growth. This essential buffering is achieved by including sodium bicarbonate-based or HEPES buffers in your cell culture media.

What is HEPES?

HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) is a zwitterionic organic chemical buffering agent. It is widely used in cell culture media to maintain stable pH levels. Although sodium bicarbonate is the most frequently used in cell culture buffer due to its nutritional benefits, the buffering capacity of sodium bicarbonate is reduced at physiological pH compared to HEPES buffer.

What is the purpose of HEPES?

HEPES is regarded as one of the best all-purpose buffers available for biological research. This is particularly important when cell cultures require extended periods of manipulation outside of a CO₂ incubator. The addition of 10–25 mM HEPES to cell culture media provides extra buffering capacity, helping to ensure the pH remains stable and protecting the cells from pH fluctuations that could otherwise impact their viability and function.

Why is HEPES in cell culture medium?

The primary purpose of HEPES is to act as a buffering agent at the physiological pH between 7.2 and 7.6, ensuring optimal conditions for cell viability. HEPES is chemically and enzymatically stable, as well as membrane impermeable, making it suitable for adding to cell culture media.

The concentration of HEPES in cell culture media may vary from 10 mM to 25 mM. In Gibco DMEM, HEPES is present at a concentration of 25 mM, while in Gibco DMEM/F-12, it is found at a concentration of 15 mM. The most commonly used concentration is 25 mM. 

Reasons to choose HEPES for your research

Gibco HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is categorized as a “Good” buffer which derives from a set of buffers described by Dr. Norman Good and his colleagues in 1966 (Good, et al. Biochemistry 1966). 

“Good buffers” such as HEPES are attributed with the following characteristics:

pKa values between 6.0 and 8.0
High solubility
Membrane impermeability
Limited effect on biochemical reactions
Very low visible and ultraviolet light absorbance
Chemically and enzymatically stable
Easy to prepare

In summary, HEPES is a reliable and effective buffering agent that enhances the stability and viability of cell cultures in various research applications. Its properties make it an excellent choice for maintaining physiological pH in cell culture media, particularly when extended manipulation outside of a CO₂ incubator is required.

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Sources:

Gibco HEPES Product Page
Buffered Saline Solutions Product Information Sheet
Cell Culture Media Supplements
Good NE, Winget GD, Winter W, Connolly TN, Izawa S, Singh RM. Hydrogen ion buffers for biological research. Biochemistry. 1966 Feb;5(2):467-77.