The Role of HEPES Buffer in Cell Culture Media
News 10 12 月, 2025
When should HEPES be used in cell culture media?
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is a commonly used zwitterionic buffer with a pKa of 7.3 at 37°C. In addition to sodium bicarbonate, HEPES can be incorporated into culture media because it is important to maintain sufficient bicarbonate for nutritional purposes. When necessary, HEPES may be added to cell culture media at a final concentration of 10–25 mM to provide additional buffering capacity. Lower concentrations are generally insufficient to control pH fluctuations, while higher concentrations can be toxic.
Because the buffering capacity of HEPES is independent of CO₂ concentration, it is ideal for maintaining pH during extended handling of cells outside of a CO₂ incubator. HEPES-buffered media can resist sharp pH changes, although it cannot completely prevent them. Due to potential cytotoxicity at high concentrations, the amount of HEPES should be reduced if toxicity is observed in specific cell lines or primary cultures.
Why use HEPES?
As cell growth and metabolism can cause significant changes in culture medium pH, appropriate biological buffers are needed to maintain a stable environment. With many available buffering agents, why is HEPES commonly used?
Most cells grow best at pH 7.2–7.4, and the effective buffering range of HEPES (pH 6.8–8.2) closely matches this requirement. Compared with PBS or TRIS, HEPES provides superior stability in maintaining medium pH. This is why HEPES is widely used in cell culture, tissue culture, protein purification, immunoprecipitation, cell lysis, live-cell imaging, and other biological or biochemical applications.
Benefits of Using HEPES
1. Stable buffer performance at low temperatures
Most biological buffers are optimized for use at around 25°C or at temperatures suitable for specific enzymes. However, when experiments require low-temperature conditions, many buffers become unsuitable. The degradation rate of HEPES increases with temperature and decreases with cooling, but its decomposition constant fluctuates far less than other buffers. This makes HEPES well-suited for maintaining enzyme structure and function at low temperatures.
2. Non-toxic to cells at appropriate concentrations
HEPES can maintain a stable pH range over extended periods. At low concentrations, it is non-toxic to cells and is commonly used in cell culture. Many bacterial, fungal, and viral media or preservation solutions include HEPES to maintain the required pH environment.