Effect of fibroblast cell which grew on amphiphilic polymer particle on the proliferation and differentiation of blood progenitor cell

Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S4–S20 have studied how to maximize the translational or secretory capacity of a host cell by engineering the secretory pathway. In this study, we improved the production of recombinant human antithrombin III (ATIII) in Chinese hamster ovary (CHO) cells. The genes encoding growth arrest and DNA damage inducible protein 34 (GADD34) and activating transcription factor 4 (ATF4), which are transcription factors involved in the unfolded protein response (UPR), were cloned from CHO-K1 cells. The production of recombinant AT-III in CHO 13D-35D cells was significantly enhanced by the overexpression of GADD34 or ATF4. The specific rate of AT-III production in the GADD34-overexpressing CHO 13D–35D cells reached approximately 28 pg/cell/day. The overexpression of GADD34 or ATF4 is a promising method of improving the production of secreted protein pharmaceuticals in CHO cells. References 1. Omasa, T., Takami T., Ohya, T., Kiyama, E., Hayashi, T., Nishii, H., Miki, H., Kobayashi, K., Honda, K., and Ohtake, H.: Overexpression of GADD34 enhances production of recombinant human antithrombin III in Chinese hamster ovary cells. J. Biosci. Bioeng. 106, 568-573 (2008). 2. Ohya, T., Hayashi, T., Kiyama, E., Nishii, H., Miki, H., Kobayashi, K., Honda, K., Omasa, T., and Ohtake, H.: Improved production of recombinant human antithrombin III in Chinese hamster ovary cells by ATF4 overexpression. Biotechnol. Bioeng., 100, 314-317 (2008).

doi:10.1016/j.jbiosc.2009.08.033

AN-P4 N-Glycan structure of humanized IgG-like bispecific antibody produced by recombinant Chinese hamster ovary cells Wook-Dong Kim, Miwako Tokunaga, Hiroyuki Ozaki, Kohsuke Honda, Takeshi Omasa, and Hisao Ohtake Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan Glycosylation is the most extensive of all posttranslational modifications and plays an important role in secretion, antigenicity, in vivo function, and the clearance of glycoproteins in blood. Glycosylation control is an important issue for the industrial production of therapeutic proteins by Chinese hamster ovary (CHO) cells. In this study, the glycosylation pattern of a humanized bispecific single-chain diabody with an Fc portion produced by recombinant CHO cells was evaluated and compared with those of a recombinant humanized anti-IL-8 antibody (IgG1) and human serum IgG. N-Linked oligosaccharide structures were estimated by twodimensional high performance liquid chromatography (2D-HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). No sialylation was observed with scDb-Fc or the anti-IL-8 antibody. From the analysis of neutral oligosaccharides, more than 90% of the N-linked oligosaccharides of scDb-Fc and the anti-IL-8 antibody were α-1,6-fucosylated. The galactosylated biantennary oligosacchrides comprise over 40% of the total N-linked oligosaccharides in both scDb-Fc and the anti-IL8 antibody. The fully galactosylated biantennary oligosaccharides from scDb-Fc and the anti-IL-8 antibody accounted for only 10% of the N-linked oligosaccharides. However, more than 20% of the Nlinked oligosacharides were fully galactosylated biantennary oligosacharides in human serum IgG. The glycosylation pattern of scDb-Fc was relatively similar to that of the anti-IL-8 antibody. Acknowledgment: This work is partially supported by grants from the NEDO of Japan, the Program for the Promotion of Fundamental

S9

Studies in Health Sciences of the NIBIO, and a grant-in-aid for Scientific Research from the JSPS. Reference 1. Omasa, T., Tanaka, R., Doi, T., Ando, M., Kitamono, Y., Honda, K., Kishimoto, M., and Ohtake, H.: Decrease in antithrombin III fucosylation by expressing GDP-fucose transporter siRNA in Chinese hamster ovary cells. J. Biosci. Bioeng., 106, 168-173 (2008).

doi:10.1016/j.jbiosc.2009.08.034

AN-P5 Effects of palindrome structure on dihydrofolate reductase gene amplification in Chinese hamster ovary cells Joon Young Park, Yasuhiro Takagi, Miyuki Yamatani, Kohsuke Honda, Takeshi Omasa, and Hisao Ohtake Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan Chinese hamster ovary (CHO) cells are widely used for the commercial-scale production of therapeutic proteins. Dihydrofolate reductase (DHFR) gene amplification is frequently used to construct productive CHO cells. However, the gene amplification mechanism is largely unknown. To elucidate the mechanism, we constructed a CHO genomic bacterial artificial chromosome (BAC) library from a mouse Dhfr-amplified CHO DR1000L-4N cell line. From the screening of high-density replica (HDR) filter hybridization and the analysis of Southern hybridization using a Dhfr probe, we selected one BAC clone (Cg0031N14) among 7 BAC clones and determined the CHO genomic DNA sequence flanking the Dhfr. The CHO genomic sequence contains a large palindrome structure and it is estimated that the structure plays an important role in Dhfr amplification. In this study, we investigated the effect of the palindrome structure derived from BAC clone Cg0031N14 on Dhfr amplification in CHO cells. We constructed expression vectors containing the palindrome structure derived from Cg0031N14 and transfected them into the CHO DG44 cell line. The transfected CHO DG44 cells could adapt quickly to the high DHFR inhibitor (methotrexate) concentrations compared with the ordinal Dhfr-transfected CHO DG44 cells. doi:10.1016/j.jbiosc.2009.08.035

AN-P6 Effect of fibroblast cell which grew on amphiphilic polymer particle on the proliferation and differentiation of blood progenitor cell Hiroshi Kunieda,1 Masaki Hiramoto,2 Wilhelm R. Glomm,3 Shin Aizawa,4 and Masahiro Yasuda1 Department of Chemical Engineering, Osaka Prefecture University, Gakuencho, Naka-ku, Sakai, 5998531 Osaka, Japan 1 Department of Metabolic Disorder, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, 1628655 Tokyo, Japan 2 Department of Chemical Engineering, Norwegian University of Science and Technology (NTNU), Sem Sælands vei 4, Trondheim N-7491, Norway 3 and Department of Functional Morphology, Nihon University School of Medicine, 30-1 ootaniguchikamimachi, Itabashi-ku, 1738610 Tokyo, Japan 4

S10

Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S4–S20

Polymer particle which has both acrylic base polymer network and epoxy grafted polymer chain form its surface was synthesized. Particle size was about 200 μm and degree of polymerization of grafted epoxy chain was ranged from 500 to 1000. When suspended fibroblast cell was added to this particle, a majority of cells were adhered on particle surface and cell number for 5 days was 4.5-fold of the primary cell. To study the effect of the particle immobilization of MS-5 cell on blood progenitor cell, human CD34 positive hematopoietic cell was obtained from cord-blood using immuno-beads system and co-cultured with MS-5 cell grown on particles. When CD34 positive cell was added to MS-5 cell grown on cell culture flask, the production of differentiated blood cells, such as granulocyte and macrophage was stopped at 8 weeks culture. However, in the case of co-cultured with MS-5 cell grown on particle, the production of differentiated blood cells maintained at 0.25-fold level at a primary culture. This result indicated that MS-5 cell on polymer particles supported in vitro the survival of hematopoietic stem cells and the granulopoietic differentiation. doi:10.1016/j.jbiosc.2009.08.036

AN-P7 Chromosome number, anatomy and gene expression of antisense ACC oxidase in transgenic Dendrobium draconis Rchb.f Sumontip Bunnag, and Thamolwan Seepor Khon Kaen University, Khon Kaen, Thailand The objectives of this research were to compare the chromosome number, anatomy and gene expression of antisense ACC oxidase between transgenic and non-transgenic of Dendrobium draconis. The experiments were conducted by culturing orchids on modified MS medium (1) containing 5 mg/l hygromycin and 15% coconut water for 4 months. It was found that the frequency of hygromycin-resistance of D. draconis were 80% while escape plants was found for 20%. To reconfirm the frequency of hygromycin-resistant plants, we cultured both selected transgenic orchids in the same medium for more 8 months. The result showed that survival rate of transgenic orchids was 100%. Using GUS assay (2) to test transformation efficiency, it was found that transgenic orchids show gus activities. An anatomical study in leaves, stems and roots of transgenic orchids showed blue stains in all tissues while non-transgenic tissues showed negative results. The successful transformation was confirmed by PCR analysis. The PCR indicated the intergration of 35S promoter, NOS terminator and antisense ACC oxidase in transgenic orchids. A cytological study in root tip cells in transgenic orchids and non-transgenic orchids showed that the chromosome number was 2n = 38. Chlorophyll content in transgenic orchids in comparison with non-transgenic orchids exhibited no significant difference. References 1. Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W.: GUS fusion: B-glucuronidase as a sensitive and versatile fusion marker in higher plants. EMBO J., 6, 3901-3907 (1987). 2. Murashige, T. and Skoog, F.: A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physilo. 15, 473-492 (1962).

doi:10.1016/j.jbiosc.2009.08.037

AN-P8 Study on mechanism of spatiotemporal formation of renal vasculature Masayuki Okada, Ai Okamoto, and Pi-chao Wang University of Tsukuba, Tsukuba, Ibaraki, Japan Whether the formation of renal vasculature was due to vasculogenesis, angiogenesis or both of them has not been clarified yet, how to elucidate the fact became urgent and development of a new technique can improve the achievement of regenerative medicine on kidney. However, the formation of renal vasculature during the development stage remains unclear because the current in vivo imaging technique cannot be applied to such kind of observation. In this study, I focus on developing an in vivo imaging technique so as to observe the vasculature in renal cortex in real time. For developing this novel technique, fluorescent contrast agent, Angiosense 680, was injected into the isoflurane anesthetized fetus C57BL/6 mice (E(embryo)16.5 ∼E18.5, P(postnatal)3), and opened for exposure of the kidney. The mice were subjected to near infrared laser microscope (IV100; Olympus) immediately. The results show that renal vasculature after E16.5 mice can be observed by using IV100. Angiosense 680 leaked from the blood vessels in E17.5 mice, while no leak could be found in E18.5 and P3 mice. This fact that high blood permeability appeared to E17.5 instead of E18.5 mice suggests that immature vasculature still existed in glomeruli and tubules in renal cortex which supplied oxygen and nutrition necessary for renal tissue formation. On the other hand, vasculature in E18.5 formed mature structure and can prevent blood permeability from capillaries of glomeruli and tubule as the roles they played in P3, and thus no blood permeability was found in E18.5 and P3 mice. This fact indicates that E18.5 may be a critical point between the immature and mature period of blood capillary in glomeruli and tubule in renal cortex which supplied oxygen and nutrition regulation. Further investigation on the blood permeability in cell–cell adhesion in blood vasculature using immunofluorescent stain will be undertaken. doi:10.1016/j.jbiosc.2009.08.038

AN-P9 The role of type V collagen in the pathological kidney Qi Zhang, Pi-Chao Wang, and Yusuke Murasawa University of Tsukuba Graduate School of Life and Environmental Sciences, Tsukuba, Ibaraki, Japan Our previous study shows that type V collagen fibrils can induce cell migration, enhance cell–cell interaction and cause ECM re-modeling on normal glomerular endothelial cell. However, its role on pathological kidney is unclear (ref. 1). In this study, we used animal model (ICR mice) with PAN (puromycin aminonucleaoside ) syndrome which produces proteinuria after intravenous injection of PAN to investigate the morphological change of kidney tissue (ref. 2). ICR mice (male, 30– 38 g) was intravenous injected with PAN (50 mg/kg), and proteinuria was confirmed by color indicator after injection. The results showed that proteinuria reached to the peak on day 5 after PAN injection. We therefore dissected mice on day 6 and prepared frozen cross-section of kidney by use of microtome. The morphological observation of mice kidney samples stained with HE and fluorescent immunochemicals was conducted by light microscope and confocal laser microscope, respectively (Figure). It was found that the expression of collagen I and IV decreased significantly