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  • 1
    Electronic Resource
    Electronic Resource
    Adaptation of cholesterol-requiring NS0 mouse myeloma cells to high density growth in a fully defined protein-free and cholesterol-free culture medium (1995)
    Springer
    Cytotechnology 17 (1995), S. 203-211 
    Details
    ISSN: 1573-0778
    Keywords: NS0 ; mouse myeloma ; cholesterol-free ; cholesterol auxotroph ; protein-free
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract NS0 has been used as a fusion partner for the production of hybridomas and has more recently been engineered to produce recombinant protein. A protein-free culture medium, designated W38 medium, has previously been developed which supported high density growth of rat myeloma and hybridoma cell lines. NS0 cells failed to grow in W38 medium and in a number of protein-free culture media which support the growth of other myeloma cell lines. NS0 cells are derived from the NS-1 cell line, which is known to require exogencus cholesterol. It was found that NS0 cells grew in W38 medium supplemented with phosphatidylcholine, cholesterol, and albumin and that NS0 were auxotrophic for cholesterol. Protein-free growth of NS0 cells was achieved by using β-cyclodextrin to replace albumin as a lipid carrier. The maximal cell density reached in this protein-free medium was in excess of 1.5×106 cell ml−1. The lipid supplements in the medium precipitated after a few days storage at +4°C. In order to overcome this problem a protocol was developed which allowed NS0 cells to be adapted to cholesterol-independent growth in W38 medium. NS0.CF (cholesterol-independent NS0 cells) were cultured continuously in W38 medium for several months. In shake flask culture a cell density of 2.4×106 cells ml−1 was achieved in W38 medium compared with 1.41×106 cells ml−1 in RPMI 1640 medium containing 10% foetal bovine serum. NS0.CF cells readily grew in a 1 litre stirred bioreactor using W38 medium supplemented with Pluronic F68 reaching a density of 3.24×106 cells ml−1. NS0.CF were cloned protein-free by limiting dilution in W38 medium, giving colonies in wells that were seeded at an average density of 0.32 cells per 200 μl. This study has demonstrated for the first time the growth of a cholesterol-requiring mouse myeloma cell line in a completely defined protein-free medium and its subsequent adaptation to cholesterol-independence.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749658
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    Paper (German National Licenses)
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  • 2
    Electronic Resource
    Electronic Resource
    The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker (1995)
    Springer
    Cytotechnology 18 (1995), S. 207-217 
    Details
    ISSN: 1573-0778
    Keywords: serum-free ; mouse myeloma ; NS0 cells ; humanised monoclonal antibody ; glutamine synthetase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A protein-free growth medium (W38 medium) had previously been developed for the NS0 mouse myeloma cell line which is cholesterol-auxotrophic. This paper describes the development of a protein-free growth medium for NS0 cells expressing humanised monoclonal antibody using GS (glutamine synthetase) as a selectable marker. Several GS-engineered NS0 cell lines expressing humanised monoclonal antibody grew in a modification of W38 medium which maintained GS-selection, supplemented with cholesterol, phosphatidylcholine and β-cyclodextrin. Further studies showed that additional glutamic acid, asparagine, ribonucleosides and choline chloride improved cell growth. Amino acid analysis identified a number of amino acids that were being depleted from the culture medium. NS0 cell lines 9D4 and 2H5 expressing CAMPATH-1H* were adapted to enable them to grow serum-free in the absence of cholesterol and β-cyclodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), supplemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86×106 cells ml−1 producing 76.6 mg l−1 of antibody. CAMPATH-1H antibody produced using serum-free medium was found to be functionally activein vitro in the Antibody Dependant Cellular Cytotoxicity (ADCC) assay.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00767768
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    Paper (German National Licenses)
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