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Physico-chemical conditions

One important factor when culturing cells in vitro is to control the Physico-chemical conditions. The culture medium is the most important component of the cell culture environment because it provides the necessary nutrients, but also because it provides other components such as sodium bicarbonate or sometimes red phenol, to help control the required Physico-chemical conditions.

pH

Most normal mammalian cell lines grow well at pH 7.4, with very little variability among different cell strains. Therefore, to control the pH of the cell culture environment some media include pH indicators such as phenol red which also protects them from UV light degradation.

CO2

The growth medium controls the pH of the culture and buffers the cells in culture against changes in the pH. Usually, this buffering is achieved by including a CO2-bicarbonate based buffer. Because the pH of the medium is dependent on the delicate balance of dissolved carbon dioxide (CO2) and bicarbonate (HCO3-), changes in the atmospheric CO2 can alter the pH of the medium. Therefore, it is necessary to use exogenous CO2, being 5% CO2 the most used concentration.

Two merged figures. The first figure shows eighteen eppendorf tubes. Each of them contains a solution of phenol red at different pH. The pH range goes from 6 to 8 and the color varies depending on its change. At pH 6 the color starts being yellow and it eventually turns red at pH 8 going through orange around pH 7. The second image is a reaction where carbon dioxide and water form carbonic acid. Carbonic acid dissociates into bicarbonate and a proton, this reaction is in equilibrium. On the other side, sodium bicarbonate dissociates into sodium and bicarbonate. The bicarbonate produced in that dissociation reaction is used in the formation of carbonic acid.

Figure 1: (1) Phenol red color variations depending on pH. (2) Balance of dissolved CO2 and HCO3- required to maintain pH.

Temperature

The optimal temperature for cell culture largely depends on the body temperature of the host from which the cells were isolated. Most human and mammalian cell lines are maintained at 36°C to 37°C for optimal growth. This is one of the reasons for pre-warming the reagents before using them, to avoid thermal shock in cells.

However, these general conditions may vary depending on the cell type. Therefore, it is important to familiarize yourself with the cell type you are working with to avoid undesired phenotypes or unsuccessful cultures.