Influence of environmental conditions on animal cell culture

5 Influence of environmental conditions on animal cell culture 

Cell culture systems must provide the physiological conditions for cell survival and proliferation. In vitro, animal cell growth is dependent on several factors, such as pH, temperature, osmolality, gas concentration (oxygen and CO2), available surface substrata, and state of the cells at inoculation (Freshney, 2005). Other factors that impact the culture are medium composition, which can differ extensively between cell lines and is discussed in detail in Chapter 5, as well as susceptibility to hydrodynamic stress, as discussed in Chapter 7. 

It is possible to monitor pH variation by adding a pH indicator. The compound most used is phenol red, which is rose-colored at pH 7.8, red at pH 7.4, orange at pH 7.0, and yellow at pH 6.5. Nevertheless, it is convenient to point out that most commercially available phenol red contains impurities that could influence cell behavior. Also, this compound can interfere with the interpretation of experimental data obtained by the use of fluorescence and absorbance techniques. 

Culture medium needs to be buffered to compensate for CO2 and lactic acid derived from glucose metabolism. Most culture media employed for animal cells are buffered with CO2 originating from the gaseous phase, in equilibrium with sodium bicarbonate (NaHCCF) added to the culture medium, as described by the following equilibrium reaction  

An increase in CO2 tends to increase H+ and HCC 3 in reaction (3), consequently increasing medium acidity. In compensation, the increase in HCC 3 causes NaHCCb formation through reaction (4), until an equilibrium is reached at pH 7.4. In summary, a medium pH decrease due to a CO2 increase in the gas phase is neutralized by the action of sodium bicarbonate, with the pH stabilizing at 7.4. Traditionally, culture media are buffered with sodium bicarbonate at a final concentration of 24 mM. 

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