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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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It should be kept in mind that the hormones that stimulate growth will not necessarily be identical to those that stimulate functional responses or differentiation. In this light, when testing for hormone effects on growth, both stimulatory and inhibitory effects should be noted. There are several possibilities of how a hormone may affect these processes:
1. Promote growth and increase or decrease function.
2. Inhibit growth and increase or decrease function.
3. Have no effect on growth but increase or decrease function.
4. Inhibit growth by promoting differentiation to a nonmitotic phenotype (Li et al., 1996b).
If the desired endpoint is to optimize a specific cell function, for example, the secretion of a specific protein, then protein secretion should be measured and used to optimize the medium supplements instead of cell number. Figure 8.1 illustrates the effect of various
Page 132
Figure 8.1.
Differential hormonal regulation of growth and prolactin secretion by GH3 cells. One can easily see that prolactin secretion and growth are regulated independently. To optimize growth one might use serum or the six-factor (6F) supplement. But for optimal prolactin secretion, one would wish to eliminate T3 from the 6F condition and supplement with Thyrotropin Releasing Hormone (TRH).
hormones on the growth and prolactin secretion of GH3 cells. Figure 8.2 illustrates the effect of various hormone combinations on survival, steroid secretion, and human Chorionic Gonadtropin (hCG) binding in primary porcine Leydig cell cultures. Figure 8.3 shows growth inhibition of neuroepithelial precursor (NEP) cell line cells by Fibroblast Growth Factor (FGF) and Transforming Growth Factor Beta (TGFb). While the growth inhibition
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Figure 8.2.
Differential hormonal regulation of survival and function in primary cultures of porcine Leydig cells. The conditions required to optimize (hCG) binding, cell number, and testosterone secretion are different. However, the maximal hCG binding seen in the insulin/ transferrin (Ins/Tf) conditions might not be the preferred condition, since this represents an increase in binding over the levels in the freshly isolated cells, while the 3H (Ins/Tf and EGF) condition maintains the receptor level found initially.
Page 133
Figure 8.3.
Differential hormonal regulation of growth and differentiation in the NEP cell line. The cells grow optimally in the conditions shown in the top photo. FGF inhibits cell growth (bottom graph), but, as seen in the middle photo, leads to a differentiation of the cells to a neuronal phenotype.
curves look similar, it is apparent from the photographs that TGF? is killing the cells, while FGF is causing differentiation to a nonmitotic neuronal phenotype (Li et al., 1996b).
These figures illustrate the above statement that the conditions for optimal growth are not necessarily those for optimal expression of function, and further, that various functions may be regulated differently. Understanding this, one can then design experiments to provide an environment that will maximize the expression of one function but potentially decrease other cell functions. Thus, "maximal" may not be "optimal" from the viewpoint of the cell, however the investigator looks at it.
Finally, the dose of hormone required for growth or function may vary from one cell line to another, from one function to another within the same cell type, or in different media or hormone supplements. This is illustrated in Fig. 8.4. TM3 cells show a 70-fold increase in growth in response to transferrin (TF), an iron transport protein, in Ham's F12 medium (with FeSO4 omitted). Half-maximal response is seen at 25 ng/ml. These cells do
Page 134


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Figure 8.4.
Growth response of several different cell lines to transferrin supplementation of defined medium. The TM4 cell line has a modest (at fivefold) growth response to transferrin, with no species specificity, while transferrin is a major growth promoter for the TM3 cell line.
not grow at low densities in this medium without TF but grow rapidly with just three factors, of which TF is the most critical. However, the Tr- cells, while sensitive (half maximal 50 ng/ml), do not have a stringent TF requirement, and grow slowly in 3H. Thus, only a fourfold increase in growth is seen with TF. The TM4 cells, in contrast, have a half-maximal response at 500 ng/ml TF. Thus, while three different cell lines may all respond to the same factor, the concentrations required and extent of growth stimulation may differ. In addition, nonhormonal components of the medium may affect cell response to a factor. Adding
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