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CHO cell culture

Zhen Lei, Abdul Sami Shaikh, Wenshuai Zheng, Xiao Yu, Jingui Yu, Jingxin Li
Itch, the unpleasant sensation that evokes a desire to scratch, accompanies numerous skin and nervous system disorders. However, the molecular mechanisms of itch are unclear. Acid sensing ion channel 3 (ASIC3) is a sensor of acidic and primary inflammatory pain. The whole-cell patch-clamp technique was used to determine the effect of Chloroquine (CQ)on ASICs currents in primary sensory neurons or the Chinese hamster ovary (CHO) cells transfected with rat-ASIC1a or- ASIC3. Site-directed mutagenesis of plasmid was performed...
October 22, 2016: Journal of Neurochemistry
Jae Gyu Yoo, Byeong-Woo Kim, Mi-Rung Park, Deug-Nam Kwon, Yun-Jung Choi, Teak-Soon Shin, Byung-Wook Cho, Jakyeom Seo, Jin-Hoi Kim, Seong-Keun Cho
Objective: The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. Methods: Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts...
October 20, 2016: Asian-Australasian Journal of Animal Sciences
Takeshi Harada, Hideki Yamamoto, Shosei Kishida, Michiko Kishida, Chihiro Awada, Toshifumi Takao, Akira Kikuchi
Wnt5b, a member of the same family of proteins as Wnt5a of which overexpression is associated with cancer aggressiveness, is suggested to be involved in cancer progression, however details remain unclarified. We analyzed biochemical properties of purified Wnt5b and the mode of secretion of Wnt5b by cancer cells. Wnt5b was glycosylated at three asparagine residues and lipidated at one serine residue, and these post-translational modifications of Wnt5b are essential for secretion. Purified Wnt5b showed Dvl2 phosphorylation and Rac activation abilities to a similar extent as Wnt5a...
October 20, 2016: Cancer Science
Lyne Jossé, Jianling Xie, Christopher Gregory Proud, C Mark Smales
Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored...
October 19, 2016: Biochemical Journal
Steven C L Ho, Esther Y C Koh, Benjamin P C Soo, Mariati, Sheng-Hao Chao, Yuansheng Yang
BACKGROUND: Methylated CpG dinucleotides in promoters are associated with the loss of gene expression in recombinant Chinese hamster ovary (CHO) cells during large-scale commercial manufacturing. We evaluated a promoter devoid of CpG dinucleotides, CpGfree, in parallel with a similar CpG containing promoter, CpGrich, for their ability to maintain the expression of recombinant enhanced green fluorescent protein (EGFP) after 8 weeks of culturing. RESULTS: While the promoters gave similar transient expression levels, CpGfree clones had significantly higher average stable expression possibly due to increased resistance to early silencing during integration into the chromosome...
October 18, 2016: BMC Biotechnology
Dongsheng Xu, Aini Wan, Jingjing Zhang, Lin Peng, Yun Chen, Yang He, Jianfeng Yang, Jian Jin
Hepatocyte growth factor (HGF) is a potent multi-functional protein that stimulates proliferation, survival, motility, scattering and differentiation during growth and development, and has been considered to be a potential therapeutic agent for the treatment of a number of intractable diseases. The aim of this study was to enhance the expression of recombinant fusion protein HSA-HGF (R494E) in CHO cells by inhibiting the intracellular ubiquitin ligase activity. The high stable expression sub-clones with different signal peptides were selected by western blot (WB) analysis and used for suspension culture...
October 18, 2016: Bioengineered
Su Jin Kang, Ji Yeon Seo, Kye Man Cho, Chang Kwon Lee, Jeong Hwan Kim, Jong-Sang Kim
A new type of doenjang was manufactured by mixing soaked soybean, koji (Rhizopus oryzae), cheonggukjang (Bacillus amyloliquefaciens MJ1-4 and B. amyloliquefaciens EMD17), and Pichia farinosa SY80 as a yeast, salt, and water, followed by fermentation with koji that was made by fermenting whole wheat with R. oryzae. The mixed culture doenjang was designed to have a more palatable flavor and stronger biological activities than the conventional product. The extract of mixed culture doenjang showed higher antioxidant activity than the commercial doenjang as evaluated by the ferric reducing antioxidant power assay although it was not significantly different from the commercial product in 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activities...
September 2016: Preventive Nutrition and Food Science
Khawaja Muhammad Imran Bashir, Man-Gi Cho
Antibiotics are routinely used in microalgae culture screening, stock culture maintenance, and genetic transformation. By studying the effect of antibiotics on microalgae growth, we can estimate the least value to inhibit growth of undesired pathogens in algal culture. We studied the effect of kanamycin and tetracycline on the growth and photosynthetic activity of two chlorophyte microalgae, Dictyosphaerium pulchellum and Micractinium pusillum. We measured CFU mL(-1) on agar plates, optical density, fluorescence yields, and photosynthetic inhibition...
2016: BioMed Research International
Rima Matsuyama, Noriko Yamano, Namiko Kawamura, Takeshi Omasa
The establishment process of high-producing Chinese hamster ovary (CHO) cells for therapeutic protein production is usually laborious and time consuming because of the low probability of obtaining stable, high-producing clones over a long term. Thus, development of an efficient approach is required to establish stable, high-producing cells. This study presents a novel method that can efficiently establish sustainably high-producing cell lines by acceleration of transgene amplification and suppression of transgene silencing...
October 12, 2016: Journal of Bioscience and Bioengineering
Nolan B Skop, Frances Calderon, Cheul H Cho, Chirag D Gandhi, Steven W Levison
Tissue engineering using stem cells is widely used to repair damaged tissues in diverse biological systems; however, this approach has met with less success in regenerating the central nervous system (CNS). In this study we optimized and characterized the surface chemistry of chitosan-based scaffolds for CNS repair. To maintain radial glial cell (RGC) character of primitive neural precursors, fibronectin was adsorbed to chitosan. The chitosan was further modified by covalently linking heparin using genipin, which then served as a linker to immobilize fibroblast growth factor-2 (FGF-2), creating a multifunctional film...
October 2016: Journal of Tissue Engineering and Regenerative Medicine
Soon-Youn Choi, Hye-Myung Ryu, Ji-Young Choi, Jang-Hee Cho, Chan-Duck Kim, Yong-Lim Kim, Sun-Hee Park
PURPOSE: High glucose stimulates peritoneal inflammation and extracellular matrix accumulation in human peritoneal mesothelial cells (HPMCs). However, the roles of Toll-like receptor 4 (TLR4) and TLR2 in high-glucose-induced inflammation and fibrosis in peritoneal dialysis (PD) remain unclear. This study aimed to evaluate the effect of high glucose on TLR2 and TLR4 expression in HPMCs and to assess their impact on peritoneal inflammatory and fibrosis markers. METHODS: Using cultured HPMCs, TLRs expression by high-glucose (50 mM) stimulation was assessed by quantitative real-time PCR...
October 8, 2016: International Urology and Nephrology
Natalie Krahn, Maureen Spearman, Markus Meier, July Dorion-Thibaudeau, Matthew McDougall, Trushar R Patel, Gregory De Crescenzo, Yves Durocher, Jörg Stetefeld, Michael Butler
Glycoengineering of mAbs has become common practice in attempts to generate the ideal mAb candidate for a wide range of therapeutic applications. The effects of these glycan modifications on the binding affinity of IgG mAbs for FcγRIIIa and their cytotoxicity is well known. However, little is understood about the effect that these modifications have on binding to the high affinity FcγRI receptor. This study analysed the effect of variable N-glycosylation on a human-llama hybrid mAb (EG2-hFc, 80 kDa) binding to FcγRI including a comparison to a full-sized IgG1 (DP-12, 150 kDa)...
October 6, 2016: European Journal of Pharmaceutical Sciences
Mee Sun Yoon, Chanh Tin Pham, Minh-Trang Thi Phan, Dong-Jun Shin, Youn-Young Jang, Min-Ho Park, Sang-Ki Kim, Seokho Kim, Duck Cho
BACKGROUND AIMS: Few studies have examined the migration pattern of natural killer (NK) cells, especially after radiation treatment for cancer. We investigated whether irradiation can modulate the expression of chemokines in cancer cells and the migration of NK cells to irradiated tumor cells. METHODS: The expression of chemokine receptors (CXCR3, CXCR4 and CXCR6) on interleukin-2 (IL-2)/IL-15-activated NK cells was assessed using flow cytometry. Related chemokine ligands (CXCL11, CXCL12 and CXCL16) in human breast cancer cell lines (MCF7, SKBR3 and MDA-MB231) irradiated at various doses were assessed using reverse transcription-polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA)...
October 6, 2016: Cytotherapy
Jisoo Shin, Jung Ho Cho, Yoonhee Jin, Kisuk Yang, Jong Seung Lee, Hyun-Ji Park, Hyung-Seop Han, Jinkyu Lee, Hojeong Jeon, Heungsoo Shin, Seung-Woo Cho
Using small interfering RNA (siRNA) to regulate gene expression is an emerging strategy for stem cell manipulation to improve stem cell therapy. However, conventional methods of siRNA delivery into stem cells based on solution-mediated transfection are limited due to low transfection efficiency and insufficient duration of cell-siRNA contact during lengthy culturing protocols. To overcome these limitations, a bio-inspired polymer-mediated reverse transfection system is developed consisting of implantable poly(lactic-co-glycolic acid) (PLGA) scaffolds functionalized with siRNA-lipidoid nanoparticle (sLNP) complexes via polydopamine (pDA) coating...
September 26, 2016: Small
Hi Chul Kim, David Shum, Hyang Sook Seol, Se Jin Jang, Ssang-Goo Cho, Yong-Jun Kwon
Although reverse transfection cell microarray (RTCM) is a powerful tool for mammalian cell studies, the technique is not appropriate for cells that are difficult to transfect. The lentivirus-infected cell microarray (LICM) technique was designed to improve overall efficiency. However, LICM presents new challenges because individual lentiviral particles can spread through the cell population, leading to cross-contamination. Therefore, we designed a cell-defined lentivirus microarray (CDLM) technique using cell-friendly biomaterials that are controlled by cell attachment timing...
October 4, 2016: Journal of Biomolecular Screening
E Lorge, M M Moore, J Clements, M O'Donovan, M D Fellows, M Honma, A Kohara, S Galloway, M J Armstrong, V Thybaud, B Gollapudi, M J Aardema, J Y Tanir
Good cell culture practice and characterization of the cell lines used are of critical importance in in vitro genotoxicity testing. The objective of this initiative was to make continuously available stocks of the characterized isolates of the most frequently used mammalian cell lines in genotoxicity testing anywhere in the world ('IVGT' cell lines). This project was organized under the auspices of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing...
October 2016: Mutation Research
Brett A Sponseller, Sandra K Clark, Jessica Gilbertie, David M Wong, Kate Hepworth, Sarah Wiechert, Prashanth Chandramani, Beatrice T Sponseller, Cody J Alcott, Bryan Bellaire, Andrew C Petersen, Douglas E Jones
Reactive intermediates contribute to innate immunity by providing phagocytes with a mechanism of defense against bacteria, viruses and parasites. To better characterize the role of CD154 in the production of reactive intermediates, we cloned and expressed recombinant equine CD154 (reqCD154) in Chinese Hamster Ovary (CHO). In co-culture experiments, CHO cells ectopically expressing reqCD154 elicited superoxide production in monocyte-derived macrophages (MDM). Collectively, our results indicate that regulation of CD154 expression plays a role in innate host defenses...
November 1, 2016: Veterinary Immunology and Immunopathology
Hitomi Shirahama, Supriya K Kumar, Won-Yong Jeon, Myung Hee Kim, Jae Ho Lee, Soon Seng Ng, Seyed R Tabaei, Nam-Joon Cho
The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics' cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction...
2016: Journal of Visualized Experiments: JoVE
Sydney A Sherman, Jack K Phillips, J Tighe Costa, Frances S Cho, Sevan R Oungoulian, John D Finan
Traumatic brain injury (TBI) is a major cause of mortality and morbidity with limited therapeutic options. Traumatic axonal injury (TAI) is an important component of TBI pathology. It is difficult to reproduce TAI in animal models of closed head injury, but in vitro stretch injury models reproduce clinical TAI pathology. Existing in vitro models employ primary rodent neurons or human cancer cell line cells in low throughput formats. This in vitro neuronal stretch injury model employs human induced pluripotent stem cell-derived neurons (hiPSCNs) in a 96 well format...
September 27, 2016: Scientific Reports
Hyun Ryu, Hee Wook Ryu, Kyung Suk Cho
The dye decolorization rate in a cell-free culture broth of the white rot fungus Trametes versicolor CBR43 was studied, including the effects of inhibitors of NaCl, Zn(II), and Cd(II) on dye decolorization activity. The maximum rates of dye decolorization in cell-free culture broth were 1410, 44.7, 41.2, and 0.19 μmol L(-1) min(-1) for Acid Blue 62, Acid Black 175, Reactive Blue 4, and Acid Red 114, respectively. The inhibition effects of NaCl, Zn(II), and Cd(II) on dye decolorization were quantitatively compared using the half maximal inhibition concentration (IC50), which indicates the concentration of an inhibitor required for 50% inhibition...
September 23, 2016: Journal of Microbiology and Biotechnology
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