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There are several approaches to defining the hormone requirements for a given cell line. The method of choice will depend on the cell line. Several possibilities are outlined below. The initial step is to obtain conditions where the cells will survive and/or grow slowly for 3"C6 days. In most cell types, this is partly a function of inoculum density. Figure 8.7 illustrates the survival of M2R melanoma cells in serum-free medium as a function of inoculum density. For this type of cell, that is, one that will attach and survive in a serum-free medium with no supplementation, it is necessary to select only the proper inoculum density and begin testing hormones for growth-promoting effects. Once the optimal hormone supplement is found, the inoculum density required for survival will decrease. In some cases the plating efficiency of a cell line in hormone-supplemented, serum-free medium will be similar to that in serum, although this is not true for all cell types. This may be due to added requirements for attachment factors or growth factors needed only initially or at higher concentrations than those needed when cells are plated at high densities. Or it may be that autocrine factors produced by the cells and necessary for their survival or growth are unstable in the absence of serum or too diluted by the large medium-to-cell-volume ratio.
Many cells, both transformed and normal, are capable of producing substances that are required for their attachment or growth. However, some cell lines (e.g., GH3) will not survive even 24 hr or will not attach to the dish in serum-free medium. For these cells several initial approaches are used to obtain the minimal survival necessary for growth factor screening:
1. Use various purified attachment factors.
2. Precoat the dish with serum.
3. Plate cells in serum-containing medium for 12"C24 hr; then remove this medium and wash the cells with serum-free medium before testing hormones.
4. Reduce serum concentrations to the point where the cells will survive but not grow.
5. Add test hormones, vitamins, growth factors, protease inhibitors, and so forth to this reduced level of serum.
6. After finding the mitogenic factors on the first-round supplement with these factors, reduce serum further and test again.
The various hormones can then be tested under these minimal conditions. When optimal conditions for growth are found, the serum (or preincubation step) can then be omitted and/or replaced with purified attachment and/or growth factors. A few general statements can be made regarding the requirements for optimal growth of cells in serum-free medium that will be of help in approaching the problem of defining serum-free media for a cell line:
1. To date, most if not all, cell lines that have been grown in serum-free media require insulin and transferrin. These two factors should be tested first and then included in all subsequent steps.
2. Most cell lines require one or more of the growth factors. These include EGF, FGF, Insulin-like Growth Factor (IGF), somatomedins, Nerve Growth Factor (NGF),
platelet-derived growth factor (PDGF), and so on (see Table 8.2). In addition, many new growth factors are being isolated each year which are specific for a certain cell type (Barnes et al., 1991).
3. One can next test other classes of factors, including:
_b. Steroids (first try each of the major classes of androgens, estrogens, and so forth, then try
metabolites within these classes)
c. Transport and binding proteins (e.g., ceruloplasmin, HDL, LDL, albumin)
d. Pituitary hormones [or pituitary extract (Roberts et al., 1990)]
e. Thyroid hormones
f. Fatty acids (complexed to albumin; liposomes) and complex lipidsjaHDL, LDL, and so forth
g. Neurotropins or cytokines
h. Protease inhibitors that are compatible with cell culture (e.g., aprotinin, a2-macroglobulin)
i. Fat-soluble vitamins such as vitamins A, D, and E
The hormone testing is best done in a stepwise fashion, testing new hormones in the presence of those found to be growth stimulatory. This is essential in some cases, as hormone effects are seldom simply additive. One cell line, shown in Fig. 8.8, shows little or
Addition of individual hormones and 3F. While addition of hormones such as EGF and progesterone individually has no effect, these factors will stimulate growth in the presence of insulin and transferrin (Tf). This type of response requires an iterative approach to optimizing media supplements.
no growth stimulation when insulin, TF, or EGF are added singly, but when all three factors are added together, the cells grow at a rate 50% of that seen in serum. In addition to these three factors, there are eight other factors that stimulate the growth of these cells in the presence of insulin, TF, and EGF, although none show any effect alone. When the cells are plated at high densities, they can grow without the addition of any factors.
Alternatively, some hormones and growth factors can stimulate growth by themselves, but their effects, when added together, cancel each other out or are inhibitory. An example of this is seen in the effect of retinoic acid and FSH on TM4 cells (Mather et al., 1980). This cell line is derived from testicular Sertoli cells. Subsequent work in this and other laboratories has shown that Sertoli cells make transferrin, (IGF-1) and (TGF-a) (an EGF-like factor) in vivo. Thus, in vitro responses reflect the in vivo environment of these cells. In addition, some growth factors may replace the requirement for other less potent mitogens. This is the case with heregulin and FGF on Schwann cell growth (Fig. 8.9) (Li et al., 1996c). In this case, one needs to determine whether receptors for both factors are present on the cell and whether both factors are produced in adjacent cells in vivo to determine whether both factors are likely to play a role in regulating the biology of the cell in vivo. It is important to remember, as stated previously, that a hormone may have a minor effect on growth but be of great importance in regulating some other function.