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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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Altering Commercial Media for Special Uses
Sometimes an addition to a commercial medium can much improve cell growth. If cells are being grown at high densities and are very lactogenic (turn the medium acidic rapidly), the addition of more glucose to the medium may improve growth and prolong viability. One can try adding a trace element mix, such as those described by Ham et al. (Hamilton and Ham, 1977; McKeehan et al., 1976, 1977). Many of these trace elements are normally provided as trace contaminants that enter the medium in water or serum. As the medium becomes more defined and the water more pure, these trace elements need to be added to the medium formulation. Sometimes an increased concentration of vitamins can be useful. Vitamin mixtures are commercially available and can be added as such. Some vitamins such as vitamin E (or a-tocopherol) or vitamin A (retinol or retinoic acid) are not added to media mixtures because of their instability, but they might be important for some cells to survive or function in vitro.
An example of this is shown in Fig. 4.4. These primary cultures of porcine Leydig cells would all die on the third day of culture unless vitamin E was added to the medium. This effect could be mimicked by other antioxidants such as vitamin C (Mather et al., 1983). While few cultures show this dramatic a dependence on antioxidants, many types of cells
Tocopherol ^ml
Figure 4.4.
The effect of vitamin E (alpha-tocopherol acetate) on the survival of porcine Leydig cells in primary culture. Vitamin E is essential if the cells are to survive beyond the third day of culture.
Page 57
Figure 4.5.
The effect of the addition of vitamin A on the phenotype of the RL-65 cell line (C). The vitamin A-supplemented cells are more tightly packed and appear smaller in phase. As can be seen in the electron micrographs, however, the (A) vitamin A treated cells are less keratinized and more columnar in shape than the (B,D) nontreated controls, and thus reflect a more normal state for this lung cell type.
benefit by having an antioxidant included in the medium. These should be made up and stored as a concentrated stock solution and added to the medium immediately before the addition of cells (see Chapter 8).
Another example of the usefulness of supplementing media with vitamins is shown in Fig. 4.5. These are RL-65 cells, a cell line derived from Clara cells in the lung (Roberts et al., 1990). It was noticed that these cells became increasingly difficult to remove from the dish at subculture when they were left for more than a few days between subcultures. The cells seemed very resistant to trypsinization, and many cells were lost on subculture. On closer examination, it was apparent that some of the cells were differentiating into a keratinized layer on top of the monolayer. This layer of keratinized cells excluded the trypsin
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Figure 4.6.
The effect of vitamin A on cell number of RL-65 cells. Cells such as those shown in Fig. 4.5B,C were counted. Even though the cells in Fig. 4.5C look more sparse than those in 4.5D, more cells are present.
and were, of course, dead. The addition of retinoic acid to these cultures prevented keratinization and helped promote more reliable growth (Fig. 4.6). Interestingly, this ability of the RL-65 cell line reflects an in vivo property of the airway epithelium, which can keratinize in states of extreme vitamin A deficiency. A different effect of retinoic acid in vitro is demonstrated by the primary rat Sertoli cell cultures. These cells are postmitotic, and thus there is no effect of retinoic acid on growth. However, as shown in Fig. 4.7, retinoic acid was a major factor in maintaining transferrin secretion by these cells in vitro (Perez-Infante et al., 1986).
? ctependem glimglgt.gn 1 iTF by RA
3.e

3.4
3.2
1 -
i
ft 2.6-
2.1-
'
2- -,........,-.......I-J
1 10 100
RA (ng/ml
Figure 4.7.
The effect of vitamin A on transferrin secretion by Sertoli cells in primary culture. In this case the same vitamin A stimulates the secretion of a specific protein in cultures of nondividing cells.
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Medium Optimization
Many scientists have devoted their careers to understanding the role of nutrient mixtures in supporting cell growth and survival in vitro. These studies have resulted in the nutrient mixtures currently published or commercially available. There is, however, still a need for more experimentation to derive the optimal media for other cell types (e.g., newly derived cell lines, human cell lines, etc.) or other culture needs (e.g., very-high-density culture, controlling differentiation through culture conditions). Optimizing the medium in which a primary culture or cell line is grown can lead to increased growth, increased protein secretion, increased viability, increased phenotypic stability, and better control of differentiation. Optimizing the nutrient mixture is an important part of this process.
The best way to optimize the nutrient mixture is to sequentially perform dose'Cresponse curves on each component, select the optimal range for each, and retest each component. This must be done as an iterative process because the ratios of the components, as well as the absolute levels, are critical in optimizing the medium. This process should be done using the desired endpoint to screen. For example, if a medium is to be optimized to achieve maximal recombinant protein secretion, then the screen should be done using protein titer as the endpoint assayed. If growth is to be optimized, then cell number is the endpoint. Medium optimized for one parameter will not necessarily be best for others. An idealized example of three different dose'Cresponse curves from a screen to optimize media for protein secretion by CHO is shown in Fig. 4.8. Note that the cells will tolerate a broad range of concentrations for some components, but will have a very narrow optimal concentration range for others. It may also be possible to substitute some components for others, for ex-
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