Hormones and growth factors

From the earlier work discussed at the 1978 Cold Spring Harbor Meeting on Cell Proliferation there has developed a broad field concerned with the study of growth factors and hormones and their effects on cell growth and differentiation. This area is considered in more detail in Chapter 2 and is of enormous clinical importance for the treatment of cancer. As well as the work on peptide hormones much work has focused on the use of MCF-7 and ZR 75.1 cell lines which were isolated from human breast tumour tissue (Lippman et al., 1977 Engel et al., 1978). These cells respond to oestrogen treatment, but the system is not as simple as first thought and may involve paracrine responses (Leake, 1988). 

As most viral diseases can now be treated by administration of antisera it is important to be able to grow batches of virus both for 

In general, the procedure is to expose cells to a range of concentrations of the drug under test for 24 h and then to test for cell viability (using, for example, neutral red which is taken up only by living cells) or total cellular protein (Goldberg and Frazier, 1989). Such tests are most easily performed in microtitre plates ( 3.2) which allow rapid quantitation of the results using a microtitre plate reader. 

An alternative method which can also be automated by the use of the Titertek supernatant harvester (see Appendix 3) involves the measurement of radioactive chromium released into the culture medium from killed cells. The harvester consists of a set of absorbent cylinders aligned so that they may be inserted into the wells of a microtitration plate (Appendix 3). Once the supernatant in the wells has been absorbed the cylinders are transferred to counting vials and the amount of radioactive chromium released from the cell monolayer is estimated. Cells take up 51 Cr sodium chromate rapidly and the excess is readily washed away by rinsing in culture medium. 

Labelling need only be for 30 min and on subsequent death of the cell more than 75% of the radioactivity is released into the supernatant (Wigzell, 1965 Hirschberg et al., 1977). 

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The mechanisms by which the growth supplements in serum-free medium act are still not understood. In order to achieve an understanding of the biochemical basis for the hormonal and growth factor requirements of animal cells, the basic mechanism of action of hormones and growth factors must be determined. The biochemical basis for the nutritional requirements of animal cells can only be determined when we have an understanding of the metabolism of the different types of animal cells. 

Cell Culture-Derived Media-Derived Protein Impurities. Immunoassays can detect low impurity levels (<1 ppm).4 The ELISA is probably one of the most sensitive analytical methods. If bovine serum is used as a media component, then testing should include ELISAs for bovine serum albumin (BSA), bovine transferrin, bovine fetuin, and bovine IgG. Often hormones and growth factors, such as insulin or insulinlike growth factor, are used as media components. ELISAs should be used to detect and quantitate these residuals in the various production steps as well as in the final product. There are commercially available antibodies to most commonly used media components. If proprietary media components are used, then the same investment in time and effort is required for the production of specific antibodies, as described above for host cell impurities. 

This class of receptors transmits signals carried by hormones and growth factors. The structure consists of an extracellular domain for binding ligands and a cytoplasmic enzyme domain. The function of kinases is to enable phosphorylation. Phosphorylation regulates most aspects of cell life. 

It has long been known that insulin ( and other hormones and growth factors) can stimulate protein biosynthesis. The signal transduction pathway linking insulin to the translation apparatus was, imtil recently, imclear. However, insight is now being gained into this mechanism (Pause et al, 1994, review Proud and Denton, 1997). 

Plants respond to many environmental stimuli, and employ hormones and growth factors to coordinate the development and metabolic activities of their tissues. Plant genomes encode hundreds of signaling proteins, including some very similar to those used in signal transductions in mammalian cells. 

Fast metabolic adjustments (on the time scale of seconds or less) at the intracellular level are generally allosteric. The effects of hormones and growth factors are generally slower (seconds to hours) and are commonly achieved by covalent modification or changes in enzyme synthesis. 

The serum in the medium is not only expensive but also can be the source of virus or mycoplasma contamination. Since the chemical nature of serum is not well defined, its contents may vary batch after batch, which can affect the result of culture. The presence of many different proteins in serum can also complicate the downstream separation processes. For these reasons, many attempts have been made to formulate serum-free media. These formulations contain purified hormones and growth factors which can substitute for serum supplements (Butler, p.ll, 1987). 

Mammalian embryos are extremely tolerant of foreign proteins while still in utero, and all substances within the developing organism are accepted as self. This is essential during development to ensure that immune responses are not raised to proteins and peptides produced during this time. Any immunological response to developmental proteins, hormones and growth factors would have disastrous results. 

Blood serum, usually bovine-derived (calf or fetal bovine), contains amino acids, growth factors, vitamins, proteins, hormones, lipids, and minerals, among other components, as indicated in Table 5.3. Besides fetal bovine serum, serum from horse (equine), and even from humans (less common) can also be used. The main functions of serum are to stimulate growth and other cellular activities through hormones and growth factors, to increase cellular adhesion through specific proteins, and to supply proteins for the transport of hormones, minerals, and lipids (Freshney, 2005). Supplementation with bovine fetal serum is performed at concentrations from 2 to 20% in volume. 

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