The pH of culture medium

The pH value of cell culture media must be balanced and stable over time.

Biological processes are extremely sensitive and influenced by the acid-base balance.

Not only does pH modulate the activity of proton-dependent transporters present on cell membranes and organelles, but it also influences the interactions, conformation and, therefore, the functionality of lipids and proteins.

Human arterial blood is kept at 7.4 (7.35-7.45). Not surprisingly, mammalian cells and tissues in vitro thrive at the same pH levels.

Most mammalian cell lines grow well at pH 7.2–7.4; anyway, these values ​​may vary.

Insect cell lines such as Sf9 and Sf21 grow optimally at pH 6.2.

Why does pH of the medium change over time during cell culture?

Sometimes, by holding the cells in culture for a certain period of time without changing the medium, you will have noticed that the medium is acidified (if it contains phenol red, this will become more orange-yellowish). Why?

In cell cultures, pH changes are an inevitable consequence of cellular metabolism.

The culture medium generally faces more or less marked acidification depending on the cell type, number and the buffer capacity of the culture medium.

Waste products (acidic metabolites) released by cells – grown too dense or grown too long in that medium – or excessive growth of contaminants -bacteria and yeasts- will also cause a decrease in pH. Cultivating in the presence of low oxygen levels can also promote the production of lactic acid by the cells, which will reduce the pH of the culture.

An excessive increase in pH can be observed when the medium is left too long in the fridge after opening or when the CO2 level in the incubator is reduced due to a technical problem.

Phenol red, also known as phenolsulfonphthalein, is commonly used as an eassily-seen pH indicator of cell culture media.

See this post to have information about phenol red (function, advantages and disadvantages).

Buffer system in cell cultures

Since biological processes are exquisitely sensitive to acid-base equilibrium, cell cultures media need to be buffered.

A buffer is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa.

A buffered solution has the characteristic that its pH of changes very little when acid or base is added, thus ensuring a good stability of the pH of the medium in which the cells grow.

The pH buffer capacity is maximum, in a closed system, when equimolar amounts of the protonated and deprotonated form of the buffer are present, which is achieved when the pH of the solution coincides with the pKa of the buffer.

A list and a discussion of the buffers that can be used in biomedical research was originally given by Good et al. (1966) and taken up by Ferguson (1980).

Nowadays, animal cell cultures are maintained at physiological pH primarily by two different buffer systems: Bicarbonate-CO₂ and HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid).

Bicarbonate-CO₂ system

In an open system, such as the organism, the bicarbonate-CO₂ system is used as the main buffer system because (despite the low pKa = 6.15), the concentrations of the acid and of the basic form of the buffer can be actively regulated by the lungs ( CO₂) and kidneys (bicarbonate) to keep the pH at an optimal level.

In cell culture, bicarbonate-CO₂ system use a matched concentration of dissolved bicarbonate with carbon dioxide gas keeped artificially at a constant level by a CO₂ incubator to control the pH of the medium.

Despite the reduced buffering capacity and the need to use an incubator to regulate the gas phase (CO₂) tension, cell culture uses the bicarbonate/CO₂ buffer still much more than any other one, due to its reduced toxicity, low price and nutritional benefits for cells.

The presence of bicarbonate ions has a physiologically important significance because it activates the essential membrane transport processes responsible for cell pH homeostasis, influencing basal intracellular pH and its sensitivity to changes in extracellular pH.

This is a physiological mechanism of signal transduction that allows the extracellular pH to modulate cellular responses. Furthermore its use avoids possible unwarranted effects that exogenous buffers may have, (see 1,2, 3  4 and 5 as an example).

BUT HOW THE BICARBONATE-CO₂ SYSTEM WORKS?

1. Carbon dioxide dissolves in the medium forming carbonic acid as it reacts with water.
2. Carbonic acid establishes an equilibrium with the HCO3- ions contained in the medium, lowering the pH.

H₂O + CO₂ ⇔ H₂CO₃ ⇔ H+ + HCO₃−

       3. The addition of HCO₃−  pushes the equation to the left, subtracting H+ and thus increasing the pH.

Therefore, depending on the selected medium (and the concentration of bicarbonate), a well-defined corresponding CO₂ tension must be used to maintain the pH at 7.4.

Some cell types, however, may prefer higher or lower bicarbonate concentrations in the media. This requires higher or lower CO2 concentrations for incubation of the cell culture medium in order to maintain a physiological pH.

Before using the medium for a given cell line, it is essential to check the correspondence between the concentration of bicarbonate in the medium to be used and the percentage of CO2 set and regulated in the incubator.

Table:  examples of widely used classical cell culture media (bicarbonate concentration and corresponding CO₂ to be setted in the incubator). 

	Eagle’s MEM Hanks’s salts	Eagle’s MEM Earle’s salts	DMEM (Dulbecco’s modified Eagle’s medium )
NaHCO3 (g/l)	0,35	2,2	3,7
NaHCO3 (mM)	4,2	26,19	44
% CO2	atmospheric	5	10

Although CO₂ is produced by growing cells, it is not produced in a high enough level when growing at a low cell density or during lag phase.

CO₂ INCUBATOR

Use of a CO₂ incubator is a good way to control the CO₂ tension and the temperature.

Calibration should be regularly performed to ensure that the correct CO₂ tension is being maintained.

It is to be noted that it is essential to maintain pH stability during the entire culture process.

For example, even a slight disturbance, such as opening an incubator door, can cause an alteration of CO₂ levels and therefore of pH. This is a common occurrence, particularly if several people use the same incubator.

Even if the door is opened for only 30 seconds, the parameters of temperature, humidity and CO₂ concentration inside the incubator vary (to realize this, just look at the display of the incubator. The CO₂ concentration immediately drops to around 0, 3% – that of the normal atmosphere), which in itself causes a shift in the pH value within the growth medium.

If the system does not neutralize it very quickly, this could have negative consequences for cell growth.

Furthermore cell manipulation outside the CO₂ incubator must be done as quickly as possible, preparing all that is necessary before withdrawing the cells from the incubator.

HEPES

HEPES may be used in addition to bicarbonate for added buffering capacity.  It is added to the media exclusively to increase buffering capacity when cell culture requires long periods of manipulation outside of a CO₂ incubator (e.g microscope visualization if the microscope chamber hasn’t the possibility of a connection to the gas line).

HEPES has a pKa = 7.3 (at 37 ° C) and this makes it a good buffer used for cell cultures.

As said above, most cells need bicarbonate in their culture medium, therefore HEPES presence as a buffer agent does not completely eliminate the need for bicarbonate in media formulations.  HEPES has no nutritional benefit to cells

When HEPES is used without exogenous CO₂, its concentration should be almost double that of bicarbonate.

HEPES must be used at concentrations of 10-25mM ; toxicity to cells can occur at a concentration over 100 mM.

If HEPES is used, the equivalent molarity of NaCl must be omitted and osmolality must be checked.

CO₂ independent cultivation medium.

• Leibovitz L-15 medium includes high piruvate (550 mg/l) in the medium. This enables cells to increase the endogenous CO₂ production, making it indipendent of exogenous CO₂

 L-15 is therefore used frequently for the trasportation of tissue explant.

• A Gibco cultivation medium independent of  CO₂ is available.

A buffer system composed of mono and dibasic sodium phosphate and β glycerophosphate regulate pH (only a small amount of NaHCO₃ is included to meet essential bicarbonate-dependent functions). No synthetic buffers (such as HEPES) are used, thus eliminating any cytotoxic effects associated with such buffer systems.