Protection against acute paraquat toxicity by ambroxol |
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Authors: | Perin L. Donnini M. Diomede L. Romano M. Tacconi M. T. Luisetti M. Salmona M. |
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Affiliation: | (1) Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 3-6-6 Asahi-machi, Machida-shi, 194 Tokyo, Japan;(2) Present address: Research Laboratories, Kyowa Medix Co. Ltd., 600-1 Minami-ishiki, Nagaizumi-cho, Sunto-gun, 411 Shizuoka, Japan;(3) Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 3-6-6 Asahi-machi Machida-shi, 194 Tokyo, Japan |
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Abstract: | An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm2 tissue culture flask, even though the cell number was above 7×105 cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, BiofermenterTM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×107 cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca2+ and Mg2+ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca2+ and Mg2+ - Tween-PBS Phosphate-buffered saline without Ca2+ and Mg2+ containing 0.05% of Tween 20 |
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Keywords: | G-CSF high density culture Namalwa KJM-1 cells optimization productivity serum-free medium |
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