References Rdu
1. Morgan JG, Morton HJ, Parker RC. Proc Soc Exp Biol Med 1950 73 1-8. 2. Eagle H. Science 1955 122 501. 3. Dulbecco R, Freeman G. Virology 1959 8 396-397. 4. Ham R. Clonal growth of mammalian cells in a chemically defined synthetic medium. Proc Natl Acad Sci 1965 53 288. 5. Moore GE, Gerner RE, Franklin HA. Culture of normal human leukocytes. J Am Acad Sci 1967 199 519-524. 6. Eagle H. Science 1959 130 432. 7. Stoker MGP, MacPherson I. Virology 1961 14 359. 8. Stoker MGP, MacPherson I. Nature...
Recombinant Thrombolytic Agents
Recombinant tissue plasminogen activator tPA was the first product from cell culture 7 . Genentech obtained the approval for tPA production from CHO cells in 1987 Table 2 . tPA is produced in a large-scale gt 10,000 L cell culture process. Although the current tPA is highly efficacious, there are several attempts to improve the pharmacokinetics of the molecule. In addition, there is a new generation of recombinant thrombolytic agents under development. Recombinant tPA from Genentech is marketed...
Electroporation
Electroporation utilizes a pulsed electric field to disrupt the voltage gradient that exists across the plasma membrane and create reversible pores that allow DNA to enter the cell 65,66 . It is a simple and rapid method. It involves a direct physical interaction between electrical field and cell membrane so that it is less cell type specific compared with other transfection methods. Electroporation parameters like peak voltage and fall time of the discharge waveform need to be empirically...
Contents
Preface . . . . iii Contributors . . . . xi 1. Cell Culture Technology An Overview 1 Sadettin S. Ozturk Introduction 1 A Brief History of Cell Culture Technology 2 Products from Cell Culture Technology 6 Future Prospects . . . . 11 References 13 2. Recombinant DNA Technology and Cell Line Development 15 Amy Y. Shen, Jana Van de Goor, Lisa Zheng, Arthur E. Reyes, and Cell Line Development Overview 15 Cell Source and Host Cell Line Selection 17 Vectors for Expression in Mammalian Cells 19 High...
FluorescenceActivated Cell SortingBased Methods
The number of clones that need to be screened in order to identify members of the population with acceptably high productivity will be determined by the ability of the chemical selection method to find high producers within the transfected population and or the availability of a rapid high throughput screen for cell line evaluation. Fluorescence-activated cell sorting FACS methods represent the potentially most elegant means of high-throughput screening. Most methods described below can be used...
The Role of Mitochondria The TCA Cycle Respiration and ATP Generation
The TCA cycle Fig. 2 is a major pathway in the central metabolism, having two main functions It provides different compounds that are precursors for the cell ana-bolism and generates most of the metabolic energy. Regarding metabolism compart-mentalization, this pathway takes place inside the mitochondria. It is also important to highlight its central role as an internal mechanism to balance the rate of glycolysis and glutaminolysis, as discussed later. The connection between TCA and glycolysis...
Selection Vector Design
When selectable markers are used to identify cells that have stably integrated the vector sequences into their genome, they are essentially being used as surrogates to identify cells that express the product. However, the linked integration of the marker gene and the product gene is not always guaranteed, and it is possible that cells which exhibit drug-resistant phenotypes, or survive in nutrient deficient media may not express the product. Initially, genes for the product and the selectable...
HIGH EFFICIENCY SELECTION OF STABLE CELL LINES Selectable Markers
In order to differentiate cells that have integrated the plasmid into the host genome, a selectable marker that allows identification and selection of those cells is required. Moreover, by physically linking the expression of the marker gene and the product gene, and using selection strategies that impair the ability to express the marker gene product relative to product gene, it is possible to stringently select for cells producing high levels of product gene. There are several types of...
The Flexibility of Animal Cell Metabolism Interaction Between Glutamine and
Up to this point the main characteristics of glucose and glutamine metabolism have been described, giving special emphasis to the apparent deregulation of the glucose and glutamine uptake rates, which takes place at higher rates than those strictly necessary for the cell functions when the cells are exposed to a nonlimiting concentration of these main nutrients. This analysis will now be completed by including account the interaction between glucose and glutamine metabolism, together with the...
Glutamine Transport
Glutamine, as mentioned previously, is the second major component of cell culture media and is the source of most of the ammonium ions generated. It is important to mention that, under the normal conditions used for cell culture 37 C , glutamine is partially decomposed in a spontaneous way at a rate of 0.2-0.6 mM day, forming pyrrolidine-carboxylic acid and ammonium ions 39 . The rest of the glutamine is incorporated into the cell by means of the different amino acid transport systems, which...
A Brief History Of Cell Culture Technology
Methods for growth and maintenance of primary cells in vitro opened the doors for cell culture technology. Early studies with embryos and fibroblasts provided tools for studying cell behavior and function in vitro. These studies also provided a tool for developing cell culture media. The cells used in those days had a limited life span unless they were transformed or were originated from tumors. The production of biologicals from cell culture evolved gradually over the last three decades.
Genetic Engineering Of Host Cell Lines To Improve Production Characteristics
One of the most notable advances in recent years has been the application of genetic engineering approaches to rationally modify specific features of mammalian host cells to improve their utility in recombinant protein expression applications. One such example is in the area of glycosylation control. A breadth of work in the last decade has demonstrated how various reactions in the glycosylation pathway can be influenced by cell culture factors, host cell selection, and protein specific...
Cell Source And Host Cell Line Selection
There are several mammalian systems capable of yielding high-level expression of recombinant proteins that are suitable for use in the manufacture of protein pharmaceuticals. Cell lines derived from rodents such as Chinese hamster ovary CHO cells, baby hamster kidney BHK cells, and lymphoma cell lines such as NSO, SP2, and YB2 0 are the most commonly used. However, there are no restrictions on the type of host cell that can be used for commercial manufacturing provided the cells 1 support...
Mcdb 131
Inorganic salts Calcium chloride CaCl2 Calcium nitrate Ca N03 2-4H20 Cupric sulfate CuS04-5H20 Ferric nitrate Fe N03 3-9H20 Ferrous sulfate FeS04-7H20 Potassium chloride KC1 Potassium nitrate KN03 Magnesium chloride MgCl2 Magnesium chloride MgCl2 anhydrous Magnesium sulfate MgS04 Magnesium sulfate MgS04, anhydrous Mangenous sulfate MnS04 HzO Ammonium molybdate NH4 6Mn7024 4HzO Nickelous chloride NiCl2 6HzO Sodium chloride NaCl Sodium bicarbonate NaHC03 Sodium meta silicate Na2Si03 9H20 Sodium...
Early Days of Cell Culture TechnologyFirst Products
Utilization of cultured cells for the production of viral vaccines was the first application of cell culture technology. Viruses for vaccine production need living cells to propagate. Embryonic chicken in eggs traditionally was used for vaccine production. Due to increased demand, alternative methods were sought and cell culture technology was the answer. The production of polio vaccine using cells grown in culture started in 1954 4 . The cells were primary monkey kidney cells grown on surfaces...
Lipofection and Polyfection
Polyfection and lipofection are the most recently developed and probably simplest transfection methodologies for gene delivery in a variety of cell types. Lipofection refers to cationic lipid-mediated gene transfer into the cells. The cationic lipids containing a quaternary amine form positively charged liposomes that interact with negatively charged DNA to form a complex. Various models have been proposed to describe how DNA and lipids interact. The most popular model, supported by in situ...
Contributors
Ron Bates Bristol-Myers Squibb, Syracuse, New York, U.S.A. Alain R. Bernard Serono Pharmaceutical Research Institute, Geneva, Switzerland Jing-xiu Bi Institute of Process Engineering, National Laboratory of Biochemical Engineering, Chinese Academy of Sciences, Beijing, P.R. China A. Burgener Department of Microbiology, University of Manitoba, Manitoba, Canada M. Butler Department of Microbiology, University of Manitoba, Manitoba, Canada Jordi Joan Cairo Department d'Enginyeria Qu mica, ETSE,...