Serum, animal cell lines

The physiological significance of the growth requirements for established animal cell lines in serum-free medium is still an unresolved matter. Cultures of... 

Antibiotics are required to prevent microbial growth consequent to accidental microbial contamination. Supplemental serum (often bovine or fetal calf serum, or synthetic serum composed of a mixture of growth factors, hormones and metabolites typically found in serum) is required as a source of the often ill-defined growth factors required by some animal cell lines. 

In spite of these hurdles, the last two decades have seen an immense leap in animal cell culture technology both at the laboratory scale as well as the industrial scale. A variety of bioreactors and instrumentation have been ingeniously been devised for the scale up and process control of animal cell cultures. Serum-free media development has considerably reduced the downstream processing costs in the recombinant protein production and purification process. The capability to induce some cell lines to lose anchorage dependence has also been an important breakthrough. 

Vitamins and lipids are often required for animal cells to grow in serum-free medium. Phosphoethanolamine and ethanolamine are key additives that facilitate the growth of the mammary tumor cell line 64024 (Kano-Sueoka and Errick, 1981). In addition, ethanolamine promotes the growth of human lymphocytes and mouse hybridoma cells. Short-term cultures of human diploid lung and foreskin fibroblasts grow in medium that includes among its supplements soybean lecithin, cholesterol, sphingomyelin, and vitamin E. 

Administration of preformed anhbodies, taken from animals, flxm pooled human serum, or flom human cell-lines is often used to treat an existing infechon (e.g. tetanus, diphtheria) or condition (venomous snake-bite). Pooled human serum may also be administered prophylachcally, within a slow-release vehicle, for those persons entering parts of the world where diseases such as hepatitis A are endemic. Such administrations confer no long-term immunity and will interfere with conciurent vaccinalion procedures. 

Once in the serum, aluminium can be transported bound to transferrin, and also to albumin and low-molecular ligands such as citrate. However, the transferrrin-aluminium complex will be able to enter cells via the transferrin-transferrin-receptor pathway (see Chapter 8). Within the acidic environment of the endosome, we assume that aluminium would be released from transferrin, but how it exits from this compartment remains unknown. Once in the cytosol of the cell, aluminium is unlikely to be readily incorporated into the iron storage protein ferritin, since this requires redox cycling between Fe2+ and Fe3+ (see Chapter 19). Studies of the subcellular distribution of aluminium in various cell lines and animal models have shown that the majority accumulates in the mitochondria, where it can interfere with calcium homeostasis. Once in the circulation, there seems little doubt that aluminium can cross the blood-brain barrier. 

In certain cases, serum (fetal bovine serum—FBS) is added to promote the growth of cells. However, the bovine spongiform encephalopathy (BSE) problem has necessitated tight control on the quality of FBS (refer to Exhibit 10.12). This increases production and downstream processing costs. For new cell lines being developed, serum-free and protein-free media are used to circumvent the possibility of virus contamination from animal sources and the variation that may arise from use of serum from animal herds. 

Clinical Applicability. The regulatory conditions for cell therapy directly refer to the cells and the materials utilized during the cultivation. The use of cell lines, especially those of animal origin (e.g. stromal cell lines in cocultivation), and the use of animal serum should carefully be reconsidered, as this will make the seeking of clinical approval a difficult task. 

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,... 

The best system for aeration and stirring of culture medium in a bioreactor must minimize shear stress without significantly reducing the oxygen transfer (KLa) and yet avoiding the generation of foam. In some systems, protectors such as serum, Pluronic F68, PEG, dextrans, lipids, and cholesterol, may have to be used to prevent shear stress. Sensitivity to shear depends on the cell line, and fortunately the lines most often utilized industrially for the production of mAbs, such as hybridomas, CHO, and BHK-21, are among the most resistant animal cells in relation to these hydrodynamic forces (Chisti, 2000 Chu and Robinson, 2001 Wu, 1995). 

Mycoplasma can be eliminated from cell culture by treatment with immune serum (Pollock and Kenny, 1963) and passage through an animal is often effective in removing mycoplasma from tumour producing cell lines. 

In monoclonal antibody purification, biological risks are primarily related to the host animal cells, but also to animal supplements for culture medium such as fetal bovine serum or pure proteins (e.g., bovine albumin, insulin, and transferrin). A special risk associated with production of antibodies with rodent cell lines is their high load of C-type particles. These particles are considered as incomplete retroviruses. The danger regarding infecting humans is not clear. Thus, the efficient separation of these particles must be guaranteed. These particles are quantified either by immunological techniques or electron microscopy. 

Clark SA Looby D (1989) Adaptation of hybridoma cell lines to grow and secrete monoclonal antibody in serum-free/ defined medium. In Spier RE, Griffiths JB, Stephenne IS Crooy PJ (eds) Advances in Animal Cell Biology and Technology for Bioprocesses, pp. 291-297. Butterworth, London. 

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