Anchorage dependent cells

Surface interactions play an important role in the ability of certain animal cells to grow and produce the desired bioproducts. An understanding of the dynamics of cell surface interactions in these "anchorage-dependent" cells (cells that function well only when attached to a surface) will be needed, for example, to improve the design of bioreactors for growing animal cells. 

By far the greatest amount of work in the literature has been done on suspended hybridoma cells but the general tendency can be transferred to anchorage-dependent cell hnes. 

Zhou M, Smith AM, Das AK et al (2009) Self-assembled peptide-based hydrogels as scaffolds for anchorage-dependent cells. Biomaterials 30 2523-2530... 

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. 

Kalogerakis, N. and L.A. Behie, "Oxygenation Capabilities of New Basket-Type Bioreactors for Microcarrier Cultures of Anchorage Dependent Cells", Bioprocess Eng., 17, 151-156(1997). 

Synthetic Spider Silk Proteins for the In Vitro Proliferation of Anchorage-dependent Cells... 

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. 

A two-dimensional micropatterned tissue can be easily obtained by utihz-ing the inherent differences in cell adhesiveness between different micropatterned photografted regions. This was attained by photoiniferter graft polymerization with a projection mask placed on an iniferter-derivatized surface. Since protein adsorption and cell adhesion are markedly suppressed on nonionic graft polymers, such as polyDMAm, any anchorage-dependent cells such as endothelial cell adhere and proliferate only on nonirradiated surfaces, resulting in the formation of a two-dimensional patterned tissue or cellular sheet (Fig. 24). 

Hepatic cells fall into a class known as anchorage-dependent cells. Therefore, colonization is in part a function of the surface area of the device. Clearly, a scaffold that provides the highest surface area in the smallest volume (offering the highest net density in cells) would be preferred. 

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... 

Kato R, Kaga C, Kunimatsu M et al (2006) Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design. J Biosci Bioeng 101(6) 485—495... 

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.,... 

When anchorage-dependent cells are immobilized by microcarriers they can be cultivated in homogeneous bioreactors (Jo et al., 1998). Furthermore, the immobilization on the carriers allows the use of other bioreactors that will be discussed in the following sections, such as fluidized-bed and packed-bed bioreactors. The microcarriers can be separated from the supernatant by sedimentation, facilitating downstream processing or cell retention in perfusion processes. 

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,... 

Griffiths JB, Cameron DR, Looby D (1987), A comparison of units process systems for anchorage dependent cells, Dev. Biol. Stand. 66 331-330. 

The choice of cell perfusion system will depend, to a certain extent, on whether the cells are capable of growing in suspension or if they need to adhere to a solid support (anchorage-dependent cells). At present there are three main types of cell perfusion system, for use with NMR. These are discussed briefly below for more detailed discussions see Gillies et al. (1986) and Seguin et al. (1992). 

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. 

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