Culture systems mammalian cells

Mammalian cell culture is more technically complex and more expensive than microbial cell fermentation. Therefore, it is usually only used in the manufacture of therapeutic proteins that show extensive and essential post-translational modifications. In practice, this usually refers to glycosylation, and the use of animal cell culture would be appropriate where the carbohydrate content and pattern are essential to the protein s biological activity, its stability or serum half-life. Therapeutic proteins falling into this category include EPO (Chapter 10), the gonadotrophins (Chapter 11), some cytokines (Chapters 8-10) and intact monoclonal antibodies (Chapter 13). 

The culture of animal cells differs from that of microbial cells in several generalized respects, which include  

Basic animal cell culture media, such as Dulbecco s modified Eagle s medium, generally contain  

Antibiotics are required to prevent microbial growth consequent to accidental microbial contamination. Supplemental serum (often bovine or foetal calf serum, or synthetic serum 

Different animal cell types display different properties pertinent to their successful culture. Those used to manufacture biopharmaceuticals are invariably continuous (transformed) cell lines. Such cells will grow relatively vigorously and easily in submerged culture systems, be they roller bottle or bioreactor based. 

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In addition to the urgent problem of capacity, manufacturers have to cope with the operating costs of production, which are increased by the need for skilled personnel and expensive media components. Another cost driver is the inherent contamination risk when using mammalian cell culture systems. All materials must be checked closely for bacterial and viral contamination, and the presence of prions and endotoxins. This affects not only the manufacturing process, but also downstream materials and even human semm albumin (HSA) used for formulations. In the end, production costs add up to 100-1000 per gram of therapeutic protein. 

A variety of mammalian cell culture systems can be used to detect mutations induced by chemical substances. The L5178Y mouse l)nnphoma line, measuring mutation at the TK locus, is preferred. TK is an important enz)une involved in DNA synthesis. Cells are exposed to the test substance at various concentrations, in the presence and absence of a metabolic activation system, for a suitable period of time, and then subcultured to assess cytotoxicity and to allow phenotypic expression prior to mutant selection. Cells deficient in TK because of a forward mutation are resistant to the cytotoxic effects of pyrimidine analogues (antimetabolites), such as trifluorothymidine (TFT). This is because the antimetabolites cannot be incorporated into cellular nucleotides and kill the cell through inhibition of cellular metabolism. After treatment, cells are grown in a medium containing TFT mutant cells can proliferate in the presence of TFT, whereas normal cells containing TK are killed. This allows the detection of an increase in mutant... 

Large-scale mutagenicity testing is already under way. More than 12,000 chemicals had been tested by early 1981, mostly in Salmonella or mammalian cell-culture systems.11 Data are accumulating rapidly, and regulatory agencies must decide what test data are most appropriate for predicting risks to human health. 

This chapter describes some of the most important and well-studied forms of post-translational modifications of proteins, which have been associated with the production of glycoproteins as biopharmaceuticals from mammalian cell culture systems. Most attention is given to glycosylation because of its general importance for the activity of all these... 

Transgenic plant systems have the potential to produce recombinant proteins on a commodity scale (Kusnadi et al., 1997) due to the low cost of growing plants and because scale-up of production simply requires sewing seeds over a greater field area. As such they offer almost unlimited scalability (Giddings, 2001). It is estimated by Kusnadi et al. (1997) that transgenic plants can produce pharmaceutical proteins at between 10 and 50-fold lower cost than microbial fermentation systems, and 1,000 times lower than mammalian cell culture systems (Hood et al., 2002). 

J. Tree, C. Riehardson, A. Fooks, J. Clegg, D. Looby, 2001. Comparison of large-scale mammalian cell culture systems with egg culture for the production of influenza virus A vaccine strains. Vaccine 19, 3444-3450. 

Despite these disadvantages, the major benefit of transgenic technology offers a means to produce proteins at a very low cost. Mammalian cell culture systems are often used for expression of recombinant human proteins (rHP), as the latter can only be obtained in a biologically active conformation when produced in such cells. However, this approach has limited production capacity and is expensive. In contrast, the production of rHP in milk of transgenic cattle is a safe and less-expensive alternative with the advantage of better protein output (Brink et al., 2000). 

MAMMALIAN CELL CULTURE SYSTEM (by Seijo Sato)... 

Birch J R, Froud S J (1994). Mammalian cell culture system for recombinant protein production. Biologicals 22 127-133. 

This similar dependence of in vivo potency and mustard reactivity on substituent electronic properties (cf Equations I and 4) has been cited (8) as evidence that the biological activity of these compounds is directly due to the rate at which they alkylate cellular DNA. However, in complex in vivo systems the possible roles of drug transport and metabolism as well as DNA repair have also to be considered. Surprisingly, there is virtually no quantitative data available on the cytotoxicity of substituted aniline mustards in mammalian cell culture systems, where the effects of substituent electronic effects on cytotoxicity and stability can be examined in the... 

Nanocapsules covered with phospholipids can be functionalized by attaching or inserting substances with particular properties, like monoclonal antibodies which can be used for targeting. Monoclonal antibodies produced in mammalian cell culture systems are becoming increasingly important as auxiliaries for the treatment of human diseases. Antibodies [65] attached to a phospholipid layer that cover radioactive nanocapsules can be transported by blood to a specific cell for binding to a tumor marker. Functionalization can also be used for self-assembly to obtain organized two- and three- dimensional stmctures [66]. 

Park, B.G., Chun, J.M., Lee, C.J., Chun, G.T. et al (2000) Development of high density mammalian cell culture system for the production of tissue-type plasminogen activator. Biotechnol Bioprocess Eng., 5, 123-129. 

There have been many reports of experiments on the use of microorganisms in studies of DNA-damaging agents. The reader is referred to other chapters in these volumes for review of these systems. In our experiments, we use human cells in vitro, and our discussion will be limited to mammalian-cell culture systems. 

The cell transformation assay uses mammalian cell culture systems to detect phenotypic changes in vitro induced by chemical substances associated with malignant transformation in vivo. Widely used cells include SHE (Isfort et al, 1996), C3H10T1/2 (Mondal and Heidelberger, 1970) and Balb/3T3 (Matthews et al, 1993). 

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