Download (direct link):
Maintaining temperature for experiments where the cells cannot be placed in the incubator, such as time-lapse photography, requires special arrangements (see, for example, Fig. 6.17). The microscope stage or other surface the dishes are resting on can be heated or the air around the culture container can be heated by enclosing the microscope stage in an incubator box (see Chapter 6). In large-scale culture, the medium can be directly heated with heating coils or in a reservoir and circulated through the culture device. The cultures can also be placed in a "warm room." These special arrangements will be discussed in the appropriate sections. In all cases, the better the temperature control, the better and more reproducibly the cells will behave.
It is also important to be sure that the heating does not lead to undue evaporation of the medium. In an incubator this is done by maintaining a saturating humidity in the air in the incubator. Air blown over cultures to keep them warm should also have saturated humidity, and cultures kept on a heated surface will work best if the cells are in a sealed flask to minimize evaporation.
Since incubators, hoods, microscopes, freezers, and other equipment in the tissue culture room generate a good deal of heat, there should be good air turnover and a good cooling system for a culture room. If the temperature in the room exceeds 37jaC by very much for any length of time, it is possible that all the cultures will be lost, since most tissue culture incubators do not cool. If nonmammalian cell lines, such as those from insects, fish, or amphibians, are being grown in a laboratory, an incubator that will cool the cultures is desirable, since these cell types must be grown at temperatures well below 37jaC.
Regulation of extracellular and intracellular pH is also essential for survival of individual mammalian cells and of animals as organisms. The pH is not only important for maintaining the appropriate ion balance, but also for maintaining the optimal function of cellular enzymes and for optimal binding of hormones and growth factors to cell surface receptors. Even transient changes in pH can alter cell metabolism and induce the production of heat-shock proteins, a process which can lead to apoptotic cell death. As with the organism, different compartments of the cells can have widely differing pHs, but the cell will maintain the appropriate pH of its subcellular compartments if the external environment is correct. This means not only providing the correct external pH but also the proper membrane components, ions, and ratio of ions, which allow the cells to maintain its internal pH through the integrity of its ion pumps and cell membranes.
Most media strive to achieve and maintain a pH between 7.0 and 7.4, with a median of 7.2. Different cell types may have an optimal pH slightly outside this range, and cells certainly differ widely in their ability to tolerate significant deviations from this level. As with temperature, slow changes in pH are tolerated better than rapid changes that cause apoptotic cell death. Most cells will tolerate a medium in the range of 6.5 to 7.8, but media much outside this range can be lethal. Even within this range, the growth rate and cellular functions vary widely as a function of pH. Cells will release cell-bound growth factors at a pH of 4"C5 and remain intact but not viable. pH 2.0 is used to fix cells for some histochemical procedures. Cell membranes are solubilized at very basic pH, leaving basement membrane and cytoskeletal components on the culture vessel surface.
The regulation of pH is done through a variety of buffering systems. Most media use a bicarbonate"CCO2 system as a major component of the buffer system. (The interaction of medium bicarbonate levels and incubator CO2 systems is discussed in Chapter 4.) Other buffers included in most media formulations are the phosphate buffers. Media may be further supplemented with complex organic buffers or serum, which when present in levels of 5"C20% (vol/vol) of the medium provide significant buffering capacity.
An understanding of the metabolism of the cells is essential to be able to derive the best method to control cell pH using the media and buffering systems available. All cells produce lactic acid and CO2 as a by-product of their energy metabolism. This is necessary for life. Generally, the faster-growing cells will produce more lactic acid than slow-growing or nongrowing cells, but there is great variability among different types of cells. In addition, there is an interdependence between medium composition and both the absolute and relative amounts of lactic acid produced by a given cell. Cells grown in low concentrations of glucose will convert a higher percentage of the glucose into macromolecules used in cell replication and less into lactic acid than the same cells grown in high concentrations of glucose. Lactic acid itself can be taken up and metabolized by some cells. Additionally, the CO2 produced by the cells will affect the pH through the bicarbonate"CCO2 buffering system, but will also be released into the atmosphere in the culture container at a rate determined by the culture vessel. Thus, a sealed flask will build up high levels of CO2, while a fermenter with air sparging will strip much of the CO2 produced by the cells out of the vessel. The actual pH in a culture at a given time is thus determined by the culture configuration, the medium the cells are grown in, the cell type, and the buffering capacity of the medium.