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Mutational establishment of mammalian cell lines suitable in industry by proton and carbon beam irradiation
Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S4–S20 In this study, we examined the expression of recombinant GA7332 using a BCTV(Beet curly top virus) vector in plants. The formation of replicating molecules was confirmed in plants by Southern hybridization analysis. Expression of recombinant GA733-2 was analyzed by RT-PCR and Western blot analysis. Plant-derived recombinant GA7332 elicited the production of specific IgG in serum by intraperitoneal immunization. The sera derived from mice immunized with recombinant GA733-2 have a specific binding activity on colorectal cancer cells. Our findings show that plant-derived recombinant GA733-2 can be used as an effective experimental immunogen for research in vaccine development. This work was supported by a grant from the Biogreen 21 project (20070401034026). References 1. Ross, A. H., Herlyn, D., Iliopoulos, D. and Koprowski, H.: Isolation and characterization of a carcinoma-associated antigen, Biochem. Biophys. Res. Commun. 135, 297-303 (1986). 2. Herlyn, D. Herlyn, M., Ross. A. H., Ernst, C., Atkinson. B. and Koprowski, H.: Efficient selection of human tumor growth-inhibiting monoclonal anti bodies, J. Immunol. Methods. 73, 157-167 (1984). 3. Göttlinger, H. G., Funke, I., Johnson, J. P., Gokel, J. M. and Riethmiiller, G.: The epithelial cell surface antigen 17-1A, a target for antibody-mediated tumor therapy: its biochemical nature, tissue distribution and recognition by different monoclonal antibodies. Int. J. Cancer. 38, 47-53 (1986).
doi:10.1016/j.jbiosc.2009.08.051
AN-P22 Cell aggregation and subsequent phenotype expression of multi-passaged human chondrocytes in rotational culture Sonoi Rie,1 Yamamoto Shinjiro,1 Hayashi Shuhei,1 Furusaki Shintaro,2 and Shioya Suteaki1 Sojo University, Kumamoto, Japan 1 and Kanto-Gakuin, Yokohama, Japan 2 As an alternative approach to cartilage repair, transplantation of tissue-engineered cartilage is a promising method. However, chondrocytes isolated from the intact tissue lose their differentiated phenotype in a conventional monolayer culture by undergoing passages. Though three-dimensional culture using pellet can modulate the chondrogenesis of dedifferentiated chondrocyte, it has a problem that the number of the pellets to be used in an apparatus is limited. In this research rotational cultures, which enable cells to aggregate, were conducted to achieve sufficient number of cell aggregates derived from cells obtained in monolayer culture. Multipassaged human chondrocytes preserving capability of cellular proliferation were used in this study. Effects of rotational speeds ranging from 0 to 120 rpm on cell aggregation and subsequent chondrogenesis were examined. The chondrogenesis was evaluated from analysis of the gene expression of collagen type II (Col II), cartilage-specific maker protein. Seventh-passaged chondrocytes of 1.0 × 105 cells/mL were inoculated into a plastic culture dish. To avoid cell attachment on the dish and promote cell aggregation, the inner bottom surface of the dish was coated with poly-(2-hydroxyethyl methacrylate), a cell attachment inhibitor. Control culture was conducted at 20% oxygen tension, which was in equilibrium with the gas phase of oxygen in air, and at 37 °C in a humidified CO2 incubator. The medium was exchanged every 7 days. The rotational speeds of more than 90 rpm resulted in cell death due to shear stress, while those of less than 60 rpm formed cell aggregates. After formation of cell aggregates the expression of Col II was observed
S15
independently of the rotational speeds. The highest viable cell densities and cell viabilities were observed at 0 rpm. Since viable cell densities in all the aggregated cultures were reduced after inoculation, effective culture conditions to enhance cell growth inside cell aggregates should be further examined for cartilage regeneration. doi:10.1016/j.jbiosc.2009.08.052
AN-P23 Mutational establishment of mammalian cell lines suitable in industry by proton and carbon beam irradiation Chida Yasuhito,1 Terada Satoshi,1 and Keiichi Takagi2 University of Fukui, Fukui, Fukui, Japan 1 and The Wakasa Wan Energy Research Center (WERC), Turuga,Fukui, Japan 2 Mammalian cell culture is widely used for biopharmaceutical production including antibody therapeutics. Since most of the cell lines used for industry are derived from cancer or malignant cells, they continue to multiply in unregulated manner. Their infinite proliferation occurs overgrowth to inhibit the production. Therefore, suitable control of the proliferation would improve the productivity. In this study, in order to establish novel mammalian cell lines whose proliferation should be spontaneously down-regulated so as not to occur the over growth, we tried to mutate the cells by proton or carbon beam irradiation. Proton and carbon beam are different in their density, the value of linear energy transfer (LET), and the value of relative biological effectiveness (RBE), which promotes us to use both of them. As an industrial cell line, CHO-DP12, producer of recombinant humanized anti-interleukin-8 antibody, was used. Dose response curves of the cell line against the proton or carbon beam irradiation were examined by colony formation assay. Viability of the cells irradiated 1 Gy of proton beam was 90%, while that with 4 Gy was 20%. Because excess irradiation dose causes multiple mutations and reduces yield of mutated cells, cells were irradiated with proton beam between 1 Gy and 3 Gy irradiation intensity to mutate the cells. Similarly, irradiation intensity of carbon beam was between 0.5 Gy and 2 Gy. For selecting the cells having desired mutation, the administration of 5-fluorouracil (5-FU) to the cells was examined. Because 5-FU would be selectively incorporated to only the cells in S-phase (DNAsynthesis), 5-FU kills the cells continuing proliferation after cells become confluent. In the presence of 1 mM 5-FU, the proliferating cells died out within 2 weeks, while in lower concentration, some of proliferating cells survived. doi:10.1016/j.jbiosc.2009.08.053
AN-P24 Magnetic manipulation device for the optimization of cell processing Hiroshi Ito,1 Ryuji Kato,1 Mina Okochi,1 and Hiroyuki Honda1,2 Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan 1 and MEXT Innovative Research Center for Preventive Medical Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan 2
doi:10.1016/j.jbiosc.2009.08.051
AN-P22 Cell aggregation and subsequent phenotype expression of multi-passaged human chondrocytes in rotational culture Sonoi Rie,1 Yamamoto Shinjiro,1 Hayashi Shuhei,1 Furusaki Shintaro,2 and Shioya Suteaki1 Sojo University, Kumamoto, Japan 1 and Kanto-Gakuin, Yokohama, Japan 2 As an alternative approach to cartilage repair, transplantation of tissue-engineered cartilage is a promising method. However, chondrocytes isolated from the intact tissue lose their differentiated phenotype in a conventional monolayer culture by undergoing passages. Though three-dimensional culture using pellet can modulate the chondrogenesis of dedifferentiated chondrocyte, it has a problem that the number of the pellets to be used in an apparatus is limited. In this research rotational cultures, which enable cells to aggregate, were conducted to achieve sufficient number of cell aggregates derived from cells obtained in monolayer culture. Multipassaged human chondrocytes preserving capability of cellular proliferation were used in this study. Effects of rotational speeds ranging from 0 to 120 rpm on cell aggregation and subsequent chondrogenesis were examined. The chondrogenesis was evaluated from analysis of the gene expression of collagen type II (Col II), cartilage-specific maker protein. Seventh-passaged chondrocytes of 1.0 × 105 cells/mL were inoculated into a plastic culture dish. To avoid cell attachment on the dish and promote cell aggregation, the inner bottom surface of the dish was coated with poly-(2-hydroxyethyl methacrylate), a cell attachment inhibitor. Control culture was conducted at 20% oxygen tension, which was in equilibrium with the gas phase of oxygen in air, and at 37 °C in a humidified CO2 incubator. The medium was exchanged every 7 days. The rotational speeds of more than 90 rpm resulted in cell death due to shear stress, while those of less than 60 rpm formed cell aggregates. After formation of cell aggregates the expression of Col II was observed
S15
independently of the rotational speeds. The highest viable cell densities and cell viabilities were observed at 0 rpm. Since viable cell densities in all the aggregated cultures were reduced after inoculation, effective culture conditions to enhance cell growth inside cell aggregates should be further examined for cartilage regeneration. doi:10.1016/j.jbiosc.2009.08.052
AN-P23 Mutational establishment of mammalian cell lines suitable in industry by proton and carbon beam irradiation Chida Yasuhito,1 Terada Satoshi,1 and Keiichi Takagi2 University of Fukui, Fukui, Fukui, Japan 1 and The Wakasa Wan Energy Research Center (WERC), Turuga,Fukui, Japan 2 Mammalian cell culture is widely used for biopharmaceutical production including antibody therapeutics. Since most of the cell lines used for industry are derived from cancer or malignant cells, they continue to multiply in unregulated manner. Their infinite proliferation occurs overgrowth to inhibit the production. Therefore, suitable control of the proliferation would improve the productivity. In this study, in order to establish novel mammalian cell lines whose proliferation should be spontaneously down-regulated so as not to occur the over growth, we tried to mutate the cells by proton or carbon beam irradiation. Proton and carbon beam are different in their density, the value of linear energy transfer (LET), and the value of relative biological effectiveness (RBE), which promotes us to use both of them. As an industrial cell line, CHO-DP12, producer of recombinant humanized anti-interleukin-8 antibody, was used. Dose response curves of the cell line against the proton or carbon beam irradiation were examined by colony formation assay. Viability of the cells irradiated 1 Gy of proton beam was 90%, while that with 4 Gy was 20%. Because excess irradiation dose causes multiple mutations and reduces yield of mutated cells, cells were irradiated with proton beam between 1 Gy and 3 Gy irradiation intensity to mutate the cells. Similarly, irradiation intensity of carbon beam was between 0.5 Gy and 2 Gy. For selecting the cells having desired mutation, the administration of 5-fluorouracil (5-FU) to the cells was examined. Because 5-FU would be selectively incorporated to only the cells in S-phase (DNAsynthesis), 5-FU kills the cells continuing proliferation after cells become confluent. In the presence of 1 mM 5-FU, the proliferating cells died out within 2 weeks, while in lower concentration, some of proliferating cells survived. doi:10.1016/j.jbiosc.2009.08.053
AN-P24 Magnetic manipulation device for the optimization of cell processing Hiroshi Ito,1 Ryuji Kato,1 Mina Okochi,1 and Hiroyuki Honda1,2 Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan 1 and MEXT Innovative Research Center for Preventive Medical Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan 2