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The Role of Biological Buffers for pH Control in Tissue Culture

All media used in tissue culture have a synthetic mixture of inorganic salts known as a ‘physiological’ or balanced salt solution.

The basic functions of this salt solution in the medium are to maintain proper pH, maintain ideal osmotic pressure, and provide a source of energy. The growth of animal cells in a nutritionally complete tissue culture medium is usually optimal when the medium is buffered at a pH in the range of 7.2 –7.4. To function most effectively, the pKa of the chosen buffer should be as close as possible to the required pH.

The most commonly used buffer in tissue culture media is sodium bicarbonate. However, this buffer has two important disadvantages:

• The pKa of sodium bicarbonate is 6.3 at 37 C which results in suboptimal buffering throughout the physiological pH range
• Carbon dioxide is released in the atmosphere which results in an increase in alkalinity, and the number of hydroxyl ions produced increases according to the amount of sodium bicarbonate added to the medium. It is possible to control this by artificially supplying carbon dioxide to the atmosphere and preventing the gas from leaving the liquid, thereby reducing the hydroxyl ion concentration in solution.

Balanced salt solutions can be divided into two types:

Those intended to equilibrate with air in a closed system at a low concentration of sodium bicarbonate (Hank's Balanced Salt Solution) and those intended to equilibrate with a gaseous phase containing approximately 5% CO 2 at a higher concentration of sodium bicarbonate (Earle’s Balanced Salt Solution Cat. No: 3-01F00-I). Earle’s Balanced Salt Solution is a much better solution because it contains a greater amount of sodium bicarbonate, but it is more difficult to use since it requires a special gaseous mixture of 5% CO 2 with 95% air to be provided by the culture medium. If this procedure is not carried out, the pH increases rapidly at normal incubation temperatures. The purple color of the medium indicates that the pH has risen, and cell growth is inhibited. An alternative method is to use a medium which produces sufficient buffering capacity but does not require 5% CO 2.

In some cases, this can be achieved by using a medium containing Earle’s salts but having the concentration of sodium bicarbonate reduced to 0.85 g /liter. An entirely different approach was devised by Leibovitz (1963). He utilized the buffering capacity of free base amino acids, omitted sodium bi-carbonate, substituted galactose for glucose and added pyruvate. The pH of his L-15 medium is approximately 7.8 which is higher than that of most other media. Since there is no production of loss of CO 2 , the pH will not rise further. This medium makes possible the growth of cells in open culture vessels without regard to the CO 2 content of the atmosphere.

Attempts have been made in recent years to find the most suitable buffer. The most commonly utilized alternative to bi-carbonate is HEPES buffer which was first described by Good, et al. (1966). It acts as a zwitterion and has proved superior to conventional buffers in comparative biological such as with cell-free preparations. It has many properties which make it ideal as a buffer to tissue culture media, principally in that it does not require an enriched atmosphere to maintain the correct pH. HEPES does not bind divalent cat ions and is soluble to the extent of 2.25 M at 0C. Note: since the DpKa / °C of 20.014, the pH reading recorded in a HEPES buffered medium will vary inversely with the temperature of the medium.

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