CO2 INCUBATOR FREE
Increased acidity in the medium is manifested by an increase in Hydrogen (H +) ions free bicarbonate ions then react with the extra H + ions to form carbonic acid “shifting the reaction to the left”, stabilising pH. The colour of the medium helps advise if cultures are at physiological pH and can assist with troubleshooting.īicarbonate buffering works through Le Chatelier’s principle. Cell culture media are generally supplemented with Phenol Red to act as a pH indicator. A yellow hue to the medium indicates the pH is too low (acidic), a purple hue shows the pH is too high (alkaline) whereas an orangey-red hue indicates the medium is of the correct physiological pH (Figure 1).įigure 1. Generally, cell culture media are also supplemented with the indicator dye Phenol Red. The bicarbonate system, however, occurs naturally in the human body and is therefore used in in vitro cell culture to mimic human and mammalian physiology, minimising toxic side effects. There are other synthetic buffers such as HEPES which work over a wider range of laboratory conditions. The bicarbonate buffering system is not the most efficient chemical system for controlling pH. Eagle’s Minimal Essential Medium (EMEM) supplemented with Earle’s Balanced Salt Solution (EBSS) and most other cell culture media are formulated with 26mM sodium bicarbonate, DMEM on the other hand has a far higher concentration of 44mM sodium bicarbonate.
Physiological pH is generally considered to be in the range of 7.2 to 7.4 for normal tissues, but some disease states (such as cancer) may require culture at a lower pH.
The amount of sodium bicarbonate (NaHCO 3) in the medium dictates the amount of CO 2 that should be used to maintain the pH. You may also have heard or read that Dulbecco’s Modified Eagle’s Medium (DMEM) requires a higher (10%) concentration of CO 2, yet most of us use 5% CO 2 with this medium and there is scant information available to advise the impact of using lower CO 2 concentrations with DMEM.ĬO 2 is not a metabolic requirement for cell cultures, its purpose is to dissolve into cell culture medium where a small proportion of it reacts with water to form carbonic acid which in turn interacts with its conjugate base (the dissolved bicarbonate ions in the medium) to control a stable physiological pH through the bicarbonate buffering system. Most cell culture laboratories will have a humidified Carbon Dioxide (CO 2) incubator set to 5% CO 2, however it is not always obvious why our cultures need this amount of CO 2 nor what the acceptable tolerances are for the ranges in CO 2 concentration. CO 2 concentration and pH control in the cell culture laboratory
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