Development of a New Protein- and Hormone-free Medium for Hybridoma Cultivation.

Toyoda, Kazuhisa; Inouye, Kuniyo

doi:10.1271/bbb1961.55.1631

Cited by 8 publications

(2 citation statements)

References 4 publications

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“…E-RDF medium was purchased from Kyokuto Seiyaku Kogyo (Tokyo. Japan) (4). Fetal calf serum (FCS) was purchased from Commonwealth Serum Laboratories (Melbourne.…”

Section: Cell Line and Mediummentioning

confidence: 99%

Enhancement of Monoclonal Antibody Production Related to Metabolic Behavior in High Density Hybridoma Culture

Saito

Nishi

Murayama

1994

Animal Cell Technology: Basic &Amp; Applied Aspects

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The influences of dissolved oxygen (DO) concentration and culture pH on specific monoclonal antibody (MoAb) productivity of hybridoma cells were examined. Perfused, fluidized-bed, porous collagen microsphere system (Verax) as high density cell culture system was used for this experiment. To compare with the data of the suspension culture using stirred-vessel perfusion culture system, we analyzed the properties of cellular metabolic behaviors by controlling environmental conditions. We used mouse-mouse hybridoma producing IgG1 type MoAb. By using the Verax system, we succeeded in long-term perfusion culture of over 300 days. We monitored the concentrations of substrates (glucose, glutamine) and metabolites (ammonia, lactate) under various DO concentrations and culture pH, and determined optimum DO concentration and culture pH. By controlling these factors, we succeeded in enhancement of monoclonal antibody productivity. I Nl'RODUCfIONMoAb is useful for biochemistry, chemical industry and medical science. For the commercial scale production of MoAb, cells must be grown efficiently at high density with high MoAb productivity. In hybridoma culture, it has been reported that environmental factors such as 00 concentration have important effects on cell growth and MoAb production (1,2,3). We have been investigating about high density culture using fluidized-bed, microsphere culture system (Verax). But there are little previous studies on the effects of environmental factors on this kind of culture systems. Therefore these experiments were carried out to demonstrate the effects of environmental factors such as DO concentration and culture pH on MoAb production. In addition,the correlations between cellular metabolic behaviors and cell growth and also MoAb production were investigated. 408 MATERIALS AND METHODS Cell Line and MediumA mouse-mouse hybridoma producing an IgGl kappa antibody was used in this work. E-RDF medium was purchased from Kyokuto Seiyaku Kogyo (Tokyo. Japan) (4). Fetal calf serum (FCS) was purchased from Commonwealth Serum Laboratories (Melbourne. Australia). Cell Culture SystemPerfusion culture was carried out with two kinds of culture systems. As the suspension culture system. we used a stirred vessel named AXELLON (2L working volume. 5L total volume) equipped with a helical screw agitator and a bubble-free oxygenation system (Kirin Brewery. Yokohama. Japan) (5). The oxygenation was performed with a coiled gas-permeable silicon tube. Perfusion culture was performed with a rotating stainless steel membrane filter system. Benchmark GX(Membrex. Fairfield. NJ. USA) and was done after 4 days. Perfusion rate was 0.5/day. E-RDF medium containing 3% FCS was used. DO concentration was controlled at 5ppm and agitation rate was kept at 40rpm.As the fluidized-bed. porous collagen microsphere system. we used Verax System One (6.7). We measured DO concentration. culture pH and cuI ture temperature at the outlet of the reactor. Cells were immo~ilized in microsphere with E-RDF containing 1% FCS. About 1X10 cells wer...

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“…E-RDF medium was purchased from Kyokuto Seiyaku Kogyo (Tokyo. Japan) (4). Fetal calf serum (FCS) was purchased from Commonwealth Serum Laboratories (Melbourne.…”

Section: Cell Line and Mediummentioning

confidence: 99%

Enhancement of Monoclonal Antibody Production Related to Metabolic Behavior in High Density Hybridoma Culture

Saito

Nishi

Murayama

1994

Animal Cell Technology: Basic &Amp; Applied Aspects

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“…In vivo and in vitro immunization regimens provided distinct advantages for unique antigens (4). Supplementation with synthetic or cell derived reagents or growth factors enhanced the survival rate of antibody-producing cells (5)(6)(7).…”

Section: Introductionmentioning

confidence: 99%

Effect of Murine Ascites on the Ability of Hybridoma Cells to Produce Antibody and Proliferate In Vitro

Lumanglas¹,

Wang²

1993

Hybridoma

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Murine ascites has been shown to contain a variety of growth promoting activities. In this study, we examined the effects of ascites fluid on the efficiency of hybridoma production and cell growth. SP2/0 mouse myeloma cells were injected into peritoneal cavities of mice and ascitic fluid was collected. Lymphocytes from mice immunized with porcine growth hormone (pGH) were fused with myeloma cells in a standard hybridization procedure. These cells were then dispensed in 96-well plates in medium containing either 2.5% ascites or 20% fetal calf serum (FCS) and cultured for few days. Supernatants from these cultures were collected and analyzed for anti-pGH antibodies. It was demonstrated that ascites-supplemented medium increased the efficiency in generating specific antibody-secreting hybridomas by 4-fold over FCS-supplemented medium. Furthermore, hybridoma cells were cultured in microtiter plate and found to proliferate in response to ascites in a dose dependent manner. This effect was abolished by prior digestion of ascites with trypsin, indicating its protein nature. B-lymphocyte related cytokines seemed less likely involved because antibodies to IL-4 and IL-6 failed to alter the stimulatory effect of ascites. Ascites was fractionated by FPLC using Superose 12 column and the active moiety was found to be a small m.w. peptide (< 1,000 dalton). Therefore, murine ascites is capable of substituting for conventional FCS in culture medium in the area of hybridoma technology.

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Hybridoma Culture in Iron-Rich Protein-Free Medium: Cell Growth, Apoptosis, and Nature of Macromolecules Recovered From the Culture Fluid

Franěk

Dolníková

Vomastek

1992

Animal Cell Technology: Basic &Amp; Applied Aspects

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Development of a New Protein- and Hormone-free Medium for Hybridoma Cultivation.

Cited by 8 publications

References 4 publications

Enhancement of Monoclonal Antibody Production Related to Metabolic Behavior in High Density Hybridoma Culture

Enhancement of Monoclonal Antibody Production Related to Metabolic Behavior in High Density Hybridoma Culture

Effect of Murine Ascites on the Ability of Hybridoma Cells to Produce Antibody and Proliferate In Vitro

Hybridoma Culture in Iron-Rich Protein-Free Medium: Cell Growth, Apoptosis, and Nature of Macromolecules Recovered From the Culture Fluid

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