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Introductionth to Cell and Tissue Culture - Jennie P.

Jennie P. Introductionth to Cell and Tissue Culture - Plenum Press, 2002.
ISBN 0-306-45859-4
Download (direct link): introductiontocellandtissueсulture2002.pdf
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Most mammalian cell cultures are grown in incubators that are set at 37jaC. This was chosen because it is the core body temperature of Homo sapiens. One might therefore expect it to be the optimal temperature for growing many cell lines derived from human tissues. However, there is no reason to expect 37jaC to be optimal for all types of human cells, and certainly not for all cells from all mammals, although most cells from warm-blooded animals will grow at this temperature. For example, the normal temperature of the human skin is well below the 37jaC of the core of the body. Thus, it might be considered more "normal" to grow human epidermal cells at a lower temperature. Likewise, the testis of all mammalian species are maintained at a temperature several degrees lower than the core body
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temperature (33jaC for humans), and spermatogenesis will not proceed normally at core body temperature. It is therefore necessary to use a lower temperature if one wishes to create an in vitro environment appropriate for studying all aspects of the process of spermatogenesis.
In contrast, many animals used to derive cell lines (e.g., cows, cats, rats, mice, and hamsters) have a body temperature significantly different than that of humans. There is no a priori reason that cell lines from these animals' tissues should be grown at 37jaC. However, if the line has been in common use for many years, the cells might be best adapted to this temperature. Insect, worm, and fish cells, on the other hand, cannot be grown at 37jaC and require significantly lower temperatures.
There are several general statements about choosing the temperature set point for the incubator. Figure 3.1 shows packed cell volume (a measure of total cell number) and protein secretion by CHO cells grown at various temperatures. Growing cells at too high a temperature is more detrimental than too low a temperature. Even fairly short exposures to temperatures as little as 3jaC above normal may lead to the death of the entire culture. Cells can tolerate temperature 10"C20 degrees below normal for fairly long periods of time, but will grow slowly, if at all, at these low temperatures. If it is done correctly, cells can of course be frozen to extremely low temperatures of "C80jaC to "C130jaC and survive for years in cryostorage. Freezing, thawing, and storage of cells are discussed in Chapter 5.
If studies are to be done comparing properties of cells grown at different temperatures, the two stock cultures should be grown at the two different temperatures for at least one subculture before setting up the comparison experiment. Abrupt changes of temperature
I 4, 11 I 1 I T II i T-1 II 1 1 r-
0 2 4, 6 fl If 14 16 10 ?0
Figure 3.1.
The effect of temperature on CHO cell growth and recombinant protein yield in vitro. The cells were counted and secreted protein measured after 4 days in culture at the indicated temperatures.
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cause many changes in the cells that may take some time to disappear. Likewise, when observing cells in the microscope, do not leave the plates out for long periods allowing them to cool to room temperature as this will affect the growth rate and other properties of the cells. Removing a few plates at a time for observation and returning them immediately after minimizes cooling and the resulting condensation that also changes the properties of the medium.
The primary mode of maintaining temperature in cell culture is the incubator. While most cell culture incubators are set up to regulate humidity and the gaseous environment, as well as control temperature, temperature control is probably the most important part of this since there are alternative methods for controlling pH and humidity. As discussed in Chapter 2, there are several commercially available types of incubators. Generally, incubators that maintain temperature by warming and circulating heated air are more responsive to temperature changes and will restore the temperature faster after the door of the incubator is opened and closed. However, in the event of a power failure or mechanical break-down, the water-jacketed incubators will maintain the temperature for several hours after power failure. The water also acts as insulation and the circulation of air only within the incubator makes maintaining a humidified atmosphere easier.
In student laboratories, if cell culture incubators are not available, to maintain humidity cells can be grown in stoppered flasks that have been equilibrated with air exhaled from the lungs through a cotton plugged pipette to maintain the 5% CO2"Cair mixture required for many media. These flasks can then be placed in a well-insulated box with a heat source and a thermostat. Many cell lines will grow quite well in these conditions in the student laboratory. The thermostat should be able to maintain the temperature set point to within 20jaC; the tighter the range, the better. It is possible to have students build an adequate incubator for a minimal amount of money using acrylic, some form of insulation, a light bulb, and a thermostat. Periodically, independently check the incubator set point with a thermometer to make sure the readout is correct.
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