Anchorage-dependent growth

Luis Roderiguez Fernandez, J., Geiger, B., Salmon, D., Ben-Ze ev, A. (1993). Suppression of vinculin expression by antisense transfection confers changes in cell morphology, motility, and anchorage-dependent growth of 3T3 cells. J. Cell Biol. 122, 1285-1294. 

Benedict, W. F., Wheatley, W. L., and Jones, P. A., 1982, Difference in anchorage dependent growth and tumorigenicities between transformed C3H/10T1/2 cells with morphologies that are or are not reverted to a normal phenotype by ascorbic acid. Cancer Res. 42 1041-1045. 

In addition to the extensively studied models examined thus far, there are numerous other examples of the effects of retinoids on both growth and differentiation of neoplastic cells. Also, the important observation has been made that for certain neoplastic cells, anchorage-independent growth in semisolid medium is several orders of magnitude more sensitive to retinoid inhibition than is anchorage-dependent growth in monolayer. These examples are discussed below. 

Contact inhibition is a characteristic of the growth of anchorage dependent cells grown on microcarriers as a monolayer. Hawboldt et al. (1994) reported data on MRC5 cells grown on Cytodex II microcarriers and they are reproduced here in Table 17.13. 

Frame, K.K. and W.S. Hu, "A Model for Density-Dependent Growth of Anchorage Dependent Mammalian Cells", Biotechnol. Bioeng., 32, 1061-1066 (1988). 

Schindler, M., Allen, M. L., Olinger, M. R., and Holland, J. F. (1985) Automated analysis and snrvival selection of anchorage-dependent cells under normal growth conditions. Cytometry 6, 368-374. 

Schindler, M., Jiang, L-W., Swaisgood, M., and Wade, M. H. (1989) Analysis, selection, and sorting of anchorage dependent cells nnder growth conditions. Methods Cell Biol. 32,423 46. 

Fugio Y, Walsh K. Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage-dependent manner. J Biol Chem 1999 274(23) 16,349-16,354. 

The growth requirements and properties of the host cell. Cells requiring foetal bovine serum will be more expensive to use than cells for which a cheaper serum source is adequate. Cells which grow in suspension are much easier to scale up to large cultures than cells which are anchorage dependent. 

Panjehpour M, Karami-Tehrani F (2007) Adenosine modulates cell growth in the human breast cancer cells via adenosine receptors. Oncol Res 16(12) 575—585 Panjehpour M, Karami-Tehrani F (2004) An adenosine analog (IB-MECA) inhibits anchorage-dependent cell growth of various human breast cancer cell lines. Int J Biochem Cell Biol 36(8) 1502—1509... 

Chiarugi, P., and Fiaschi, T. 2006. Redox signalling in anchorage-dependent cell growth. Cell Signal. 19 672-682. 

Animal cells may be anchorage-dependent. Cells that depend on a solid substratum for growth are named adherent cells. These cells normally proliferate in monolayers and show contact inhibition, with the maximum cell yield generally limited by the available surface of the culture vessel. The yield of cells in suspension is not dependent on a solid substratum. 

As previously stated, ammonium in cell culture medium is the product of glutamine metabolism and its spontaneous decomposition at 37°C. Negrotti et al. (1989) showed that the half-life for glutamine at 37°C and pH 7.2 is only 7 days. The effects of ammonium on cell metabolism are observed from concentrations as low as 2 mM, which is easily reached in culture systems (Table 4.2). For example, the spontaneous decomposition of glutamine can result in 0.1 mM ammonia per day (Butler and Spier, 1984). Anchorage-dependent cells grown on microcarriers produce between 2 and 3 mM ammonia after growth in a batch culture (Butler et al.,... 

Another important aspect involved in the selection of transfected lines is the capacity to grow without physical support, since the scale-up of such processes is much simpler than those designed for growth of anchorage-dependent cells. Thus, cells that grow naturally in suspension are preferred, such as myeloma cells (Sp2/0 and NSO), or others that can be easily adapted to this form of cultivation, such as CHO and BHK (Chu and Robinson, 2001). 

Traditionally, the production of mAbs uses complex culture media containing glucose and amino acids as the main sources of carbon for cell metabolism, as well as vitamins, micronutrients and sometimes animal serum, usually fetal bovine serum. Chapter 5 provides a discussion on composition of culture media and recent trends in the search for formulas that do not require the use of animal serum, or of proteins of animal origin. These serum-free formulations use substitutes such as peptones, epithelial and fibroblast growth factors, hydrolysates, yeast extract, choline, and inositol. For the production of mAbs, various serum-free formulas are available, some of these developed specifically for a given cell line (Chu and Robinson, 2001). The development of those media is easier for non-anchorage-dependent cells, such as those used for mAb production. Thus, approximately 50% of the antibodies for therapeutic use are already produced using serum-free media. In some circumstances, the elimination of serum should be accompanied by the addition of other substances with the same shear stress protective effect of serum proteins,... 

Anchorage-dependent cells can be grown on solid or macroporous microcarrier beads and these can then be superfused with nutrient medium in the spectrometer. The advantages of the system include direct contact between the perfusate and the cells (see below) and growth of the cells at rates comparable to those found in tissue culture dishes. A disadvantage, with solid beads, is the relatively low cell density obtainable due to the large sample volume occupied by the beads. 

However, as described in 3.2, the traditional vessels used for growing anchorage-dependent cells have limited potential for scale-up which is expensive and cumbersome and is associated with difficulties in sampling and growth control. The realisation of such problems has led to the development of microcarriers. 

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