Download (direct link):
Cells grown in dishes in incubators are passively controlled by the type of dish, medium composition and depth, and the buffers, while cells grown in fermenters or instrumented spinners have active control of pH through the programmed addition of acid or base to maintain the pH at a given set point. This control comes at a cost, however. These added
acids and bases result in an increase in salts in the medium and a resulting increase in osmolality, which eventually will become, of itself, limiting for cell growth.
The main buffering system in most media is the CO2"Cbicarbonate system. The interaction of CO2 derived from the cells or the atmosphere with water leads to a drop in pH described by the equation below:
H?0 - CO, = H^COj = H+ 4- HCO-
Increasing the bicarbonate concentration neutralizes the effect of increased CO2 due to the following: NaHCO3 = Na+ + HCO+3. The increased H drives the equation above to the left until equilibrium is
reached at pH 7.4 (if the correct bicarbonate-CO2 ratios are used). The effect on cell growth of altering the pH by mismatched CO2-bicarbonate concentrations is shown in Fig. 3.2.
High concentrations of bicarbonate in media formulations require higher CO2 percentages in the air supply to provide an appropriate pH in the medium but also to provide greater buffering capacity. The carbonate ions themselves also affect cell function to some extent, and some cell types require bicarbonate to grow, independent of its requirement as a buffer. The choice of buffering system will clearly affect both the pH and the osmolality of the final medium, as well as other aspects of cell physiology controlled by the ionic environment.
The bicarbonate buffering system, while inexpensive and well understood, may be supplemented with a variety of organic buffers. The most widely used zwitterionic buffer is HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid). Since these organic buffers are insensitive to the CO2 level, they provide a good backup system for rapidly metabolizing cells that produce a lot of CO2, and can stabilize pH swings that occur in CO2"Cbicarbonate-buffered media when removing cells from the incubator for observation under
1.2 2 4 3.7
Match the CO2 with the correct bicarbonate level for optimal growth. The figure shows the growth of cells at different bicarbonate levels in a 5% CO2 incubator. The combination of 1.2 g/liter bicarbonate and 5% CO2 provides a pH of 7.2. Increased bicarbonate will raise the pH.
the microscope. This is particularly important when eliminating serum that has a significant buffering capacity itself. Most cells tolerate HEPES buffer added in the range of 10 to 50 mM, or even higher. However, since this is a rather expensive medium additive, it is generally used at 10"C20 mM (see Medium Preparation in Chapter 4 for preparing HEPES-buffered medium). Note that many buffers, such as Tris [tris(hydroxymethyl)aminomethane], commonly used in cell-free biochemistry are toxic to cells.
The osmolality of the medium used is determined by the medium formulation. Salts and glucose are the major contributors to the osmolality of the medium, although amino acids may also contribute significantly. Altering the osmolality significantly (by more than 50 mOsm) will almost always affect cell growth and function in some manner. This should be taken into consideration when studying the effects of the addition of ions, energy sources, or large changes in amino acid levels on cell growth and function. Almost all commercial media are formulated to have a final osmolality of around 300 mOsm. While different cells might have somewhat different iso-osmotic points, and therefore a different optimal osmolality for growth (Waymouth, 1970) or a specific function, most cells grow well in the range of 290 to 310 mOsm.
One way to confirm whether media have been properly prepared is to check the osmolality. Significant deviations outside the range of optimal osmolality will result in loss of membrane integrity, as the outside osmotic pressure becomes too much higher or lower than that which must be maintained inside the cell. In short, the cells explode or collapse. This phenomenon can be observed in minutes by removing medium from a dish of cells, replacing it with water, and observing under a microscope. If you wish to study the effects of osmolality on your cells, make up medium without sodium chloride. Add various concentrations of NaCl around the concentration that is normal for the medium you are using and determine the effect on the parameter to be studied (e.g., growth). The effect of high osmolality on cell growth and cell volume is shown in Fig. 3.3.
The effect of increasing osmolality on cell growth. The optimal osmolality for the growth of these CHO cells is approximately 290 mOsm.
Osmolality can be checked directly with an osmometer. One can also increase the osmolality of medium by adding sodium chloride. Adding 0.0292 g/liter of NaCl will raise the osmolality 1 mOsm (using a 5 M NaCl solution 1 ml/liter will raise the osmolarity 10 mM). This is a rough estimate but useful if a quick experiment is to be performed to measure the effect of increasing osmolality. Remember, in media other components such as sugars, other ions, and amino acids also contribute to the final osmolarity of the medium. Also, be aware that changing osmolality by adding NaCl alters the Na:K ratio, and this may affect other aspects of cell function, including membrane transport. Osmolality may also be increased by adding "inert," that is, nonmetabolizable, sugars such as xylose. While available medium formulations should be optimal for growing most of the commonly used established cell lines, it is advisable to keep the possibility of altering osmolality in mind when trying to culture cell types that are not commonly cultured.