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  • 1
    Electronic Resource
    Electronic Resource
    Animal cell cultivation for production of biological substances with a novel perfusion culture apparatus (1983)
    Springer
    Methods in cell science 8 (1983), S. 167-171 
    Details
    ISSN: 1573-0603
    Keywords: perfusion culture ; Namalva cells ; lymphoblastoid cells ; cell sedimentation column ; serum free medium ; interferon
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Specific standard methods of a new perfusion culture are described for growth and maintenance of mammalian cells in suspension culture at high density. The new system of perfusion consists of a suspension vessel containing a cone-type cell-sedimentation column as cell separator and serving as well for axis of impeller. In this report, Namalva cells (human lymphoblastoid cells), adapted to a serum-free medium, have been grown in this perfusion system. The cultured cells and the conditioned medium were separated in the cell-sedimentation column; cell-free expended medium was drained from culture vessel to effluent vessel. The cell-sedimentation column device has been used for the propagationin vitro of Namalva cells to densities 7 to 10 × 106 cells/ml in serum-free medium supplemented with insulin, transferrin, sodium pyruvate, selenious acid, and galactose.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01665880
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  • 2
    Electronic Resource
    Electronic Resource
    Efficient expression of human beta-interferon in Namalwa KJM-1 cells adapted to serum-free medium by a dhfr gene coamplification method (1990)
    Springer
    Cytotechnology 4 (1990), S. 173-180 
    Details
    ISSN: 1573-0778
    Keywords: beta-interferon ; dihydrofolate reductase ; gene coamplification ; Namalwa KJM-1 ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We previously reported the expression of human beta-interferon (β-IFN) (Miyajiet al., 1989) and human lymphotoxin (Miyajiet al., 1990) in Namalwa KJM-1 cells adapted to serum-free medium. To establish an efficient gene expression system, a dihydrofolate reductase (dhfr) gene coamplification method was applied to this cell line. A β-IFN expression plasmid was introduced with a dhfr expression plasmid into KJM-1 and methotrexate (MTX)-resistant derivatives were selected by a stepwise increase of MTX concentration. Among them, derivatives which showed higher expression levels of β-IFN than that achieved by the parental transformants were obtained, suggesting that a dhfr gene coamplification method can be used for efficient expression of foreign genes in KJM-1 which contains endogenous dhfr genes. Then, an improved β-IFN expression vector was constructed, which contains a dhfr transcription unit. This plasmid was introduced into KJM-1 and then, MTX-resistant derivatives were selected. Among them, the highest producer, clone 40-10-24, secreted β-IFN at a level as high as 5 μg/ml, which is about 100-fold higher than that obtained by the G418-resistant parental transformants. In addition, β-IFN produced by recombinant KJM-1 cells had the same molecular weight of that produced by fibroblasts.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00365098
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  • 3
    Electronic Resource
    Electronic Resource
    Optimization of cell culture conditions for production of biologically active proteins (1991)
    Springer
    Cytotechnology 5 (1991), S. 17-34 
    Details
    ISSN: 1573-0778
    Keywords: genetic engineering ; Namalwa cells ; perfusion culture ; scaling-up ; serum-free medium ; stable production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract We investigated the basic technology of cell culture conditions for production of useful substances such as cytokines, and related proteins produced by Namalwa cells. Namalwa cells (Klein, 1972), human B lymphoblastoid cells, were used for large scale production of alpha-interferon (Klein, 1979). Namalwa KJM-1, a subline of Namalwa cells, adapted to serum- and albumin-free medium, can grow at a high density above 1 × 107 cells/ml in suspension mode by the use of a perfusion culture system, Biofermenter™, containing a cone-type cell-sedimentation column as cell separator (Sato, 1983). Several kinds of cytokine cDNA can be introduced and expressed in Namalwa KJM-1 cells (Miyaji, 1990a,b,c). Some of these were produced in large quantities by use of a gene amplification method with dhfr (Miyaji, 1990c), even though the Namalwa KJM-1 cells contained endogenous dhfr genes. For stable production of the target protein, Namalwa KJM-1 cells are very useful host cells, because they have no effective endogenous protease activity in the conditioned medium. Using Biofermenter with micro-silicone fibers and a dialysis system, the specific productivity of the target proteins was not depressed at a high cell density.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00573878
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  • 4
    Electronic Resource
    Electronic Resource
    Establishment of Namalva cell lines which grow continuously in glutamine-free medium (1988)
    Springer
    Cytotechnology 1 (1988), S. 151-158 
    Details
    ISSN: 1573-0778
    Keywords: adaptation ; ammonia ; glutamine-free ; glutamine synthetase ; Namalva cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Glutamine has been shown to be a preferred energy source for some established cell lines and cancer cells in culture (Kovacevic, 1971; Kovacevic, 1972; Lavietes, 1974). Empirically, glutamine is the most abundant amino acid in most of the culture media developed. The major end product of glutamine metabolism is ammonia. Ammonia build up is one of the limiting factors in the proliferation of mammalian cells in higher density culture and is directly related to the initial glutamine concentration. The susceptibility of glutamine to thermodecomposition prevents the heat sterilization of glutamine-enriched media and this significantly increases the cost of medium preparation at the industrial scale. In an attempt to overcome these drawbacks, a population of Namalva cells capable of growing in glutamine-free media was established. The adapted cells were found to contain a higher level of glutamine synthetase activity which enable them to synthesize sufficient amounts of glutamine for their growth.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00146816
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  • 5
    Electronic Resource
    Electronic Resource
    Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium (1990)
    Springer
    Cytotechnology 4 (1990), S. 39-43 
    Details
    ISSN: 1573-0778
    Keywords: lymphotoxin ; Namalwa KJM-1 ; recombinant DNA technology ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a good host cell line for recombinant DNA technology. We previously reported the expression of human β-interferon (β-IFN) in Namalwa KJM-1 (Miyaji, 1989a). The utility of Namalwa KJM-1 for expression of foreign genes was further examined. As a target gene to be expressed, human lymphotoxin (hLT) cDNA was used. It was engineered for expression in Namalwa KJM-1 using a simian virus 40 (SV40)-based expression vector pAGE107 (Miyaji, 1989a). It contains all components necessary for the expression of cDNA in mammalian cells. The expression vector was introduced into Namalwa KJM-1 by electroporation. Among the transformants, clone 7 was further examined for the expression of hLT in serum-free medium. The production level of hLT was augmented with the increase of the cell density. Thus it was further indicated that Namalwa KJM-1 is useful for production of foreign gene products.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00148809
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  • 6
    Electronic Resource
    Electronic Resource
    Expression of human beta-interferon in Namalwa KJM-1 which was adapted to serum-free medium (1990)
    Springer
    Cytotechnology 3 (1990), S. 133-140 
    Details
    ISSN: 1573-0778
    Keywords: beta-interferon ; electroporation ; Namalwa KJM-1 ; recombinant DNA technology ; serum-free medium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a very useful host cell line for recombinant DNA technology. Thus, the utility of Namalwa KJM-1 for expression of foreign genes was examined. As a model system human beta-interferon (β-IFN) gene was engineered for expression in this cell line. For construction of the β-IFN expression vector pSE1β1–4, the expression vector pAGE107 was constructed and used. It contains simian virus 40 (SV40) early promoter, the rabbit β-globin RNA processing signals for splicing and polyadenylation, and SV40 early RNA processing signal for polyadenylation. In addition to the above transcription unit, pAGE107 contains the ampicillin-resistance gene and G418-resistance gene. They can confer ampicillin resistance to Escherichia coli (E. coli) and G418 resistance to animal cells. To introduce plasmid DNA into cells, electroporation is a useful method (Wong, 1982; Potter, 1984). We have established conditions for DNA-mediated transfection of Namalwa KJM-1 cell line by electroporation. Among pSE1β1–4-introduced cells, clone 1–3 was further examined for the expression of β-IFN in serum-free medium. The production level of β-IFN was elevated with the increase of the cell density. The results indicated that the Namalwa KJM-1 cell line is useful for production of foreign gene products.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00143675
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