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Cell Culture Medium (cell + culture_medium)
Selected AbstractsSTED Microscopy to Monitor Agglomeration of Silica Particles Inside A549 Cells,ADVANCED ENGINEERING MATERIALS, Issue 5 2010Sabrina Schübbe The widespread use of engineered nanomaterials increases the exposure of the materials to humans. Therefore, it is necessary to know how these materials interact with cells. One approach is to trace particles by fluorescent labeling. The aim of the present work was to study the behavior of silica particles in A549 cells. For the first time, we applied stimulated emission depletion (STED) microscopy for this approach. Therefore, SiO2 particles conjugated with Atto647N were prepared by L -arginine-catalyzed hydrolysis of tetraethoxysilane. The Atto647N labeled SiO2 particles exhibit a mean size of 128,±,7,nm and a zeta-potential of ,11,mV in cell culture medium. STED microscopy enables subdiffraction resolution imaging of single Atto647N labeled SiO2 particles not only in pure solution but also in a cellular environment. To visualize Atto647N labeled SiO2 particles inside A549 cells, the membrane was labeled and image stacks, that give three-dimensional information, were taken after 5, 24, and 48 h exposure of particles to cells. During this incubation period, an increase in particle uptake was observed and STED micrographs allowed us to evaluate the agglomeration of Atto647N labeled SiO2 particles inside A549 cells. Our results show that STED microscopy is a powerful technique to study particles in a cellular environment. [source] Superparamagnetic Hyperbranched Polyglycerol-Grafted Fe3O4 Nanoparticles as a Novel Magnetic Resonance Imaging Contrast Agent: An In Vitro AssessmentADVANCED FUNCTIONAL MATERIALS, Issue 16 2009Liang Wang Abstract Hyperbranched polyglycerol-grafted, magnetic Fe3O4 nanoparticles (HPG-grafted MNPs) are successfully synthesized by surface-initiated ring-opening multibranching polymerization of glycidol. Reactive hydroxyl groups are immobilized on the surface of 6,9,nm Fe3O4 nanoparticles via effective ligand exchange of oleic acid with 6-hydroxy caproic acid. The surface hydroxyl groups are treated with aluminum isopropoxide to form the nanosized macroinitiators. The successful grafting of HPG onto the nanoparticles is confirmed by infrared and X-ray photoelectron spectroscopy. The HPG-grafted MNPs have a uniform hydrodynamic diameter of (24.0,±,3.0) nm, and are very stable in aqueous solution, as well as in cell culture medium, for months. These nanoparticles have great potential for application as a new magnetic resonance imaging contrast agent, as evidenced by their lack of cytotoxicity towards mammalian cells, low uptake by macrophages, excellent stability in aqueous medium and magnetic fields, and favorable magnetic properties. Furthermore, the possibility of functionalizing the hydroxyl end-groups of the HPG with cell-specific targeting ligands will expand the range of applications of these MNPs. [source] Electrosprayed Enzyme Coatings as Bioinspired Alternatives to Bioceramic Coatings for Orthopedic and Oral ImplantsADVANCED FUNCTIONAL MATERIALS, Issue 5 2009Lise T. de Jonge Abstract The biological performance of orthopedic and oral implants can be significantly improved by functionalizing the non-physiological metallic implant surface through the application of biologically active coatings. In this paper, a cost-effective alternative to traditional biomedical coatings for bone substitution through exploitation of the specific advantages of the electrospray deposition technique for the immobilization of the enzyme alkaline phosphatase (ALP) onto the implant surface is presented. Since ALP increases the local inorganic phosphate concentration required for physiological mineralization of hard tissues, ALP coatings will enable enzyme-mediated mineralization onto titanium surfaces. To evaluate the bone-bioactive capacity of the ALP-coated titanium surface, soaking experiments are performed. Although the purely inorganic so-called simulated body fluid is the standard in vitro procedure for predictive studies on potential bone bonding in vivo, an alternative testing solution is proposed that also contains organic phosphates (cell culture medium supplemented with the organic ,-b; -glycerophosphate (,-b; -GP) and serum proteins), thereby resembling the in vivo conditions more closely. Under these physiological conditions, the electrosprayed ALP coatings accelerated mineralization onto the titanium surface as compared to noncoated implant material by means of enzymatic pathways. Therefore, this novel approach toward implant fixation holds significant promise. [source] REVIEW ARTICLE: An improved manufacturing process for Xyntha/ReFacto AFHAEMOPHILIA, Issue 5 2010B. KELLEY Summary., ReFacto® Antihemophilic Factor is a second-generation antihaemophilia A product manufactured using a process that includes therapeutic grade human serum albumin (HSA) in the cell culture medium, but is formulated without HSA as a stabilizer. Even though this second-generation antihaemophilia product has a good safety profile, a programme was implemented to eliminate all animal- and human-derived raw materials from the production process, thus producing a third-generation product. To that end, HSA has been removed from the master and working cell banks and from the culture medium. The hybridoma-derived monoclonal antibody formerly used in the purification process has been replaced by a chemically synthesized affinity peptide, and a virus-retaining filtration step has been added to enhance the clearance of large viruses, such as retroviruses. The purification process has been validated for the removal of a panel of model viruses and provides significant clearance of all viruses tested. Host cell- and process-derived impurity removal validations also were conducted, including host cell DNA and protein, in addition to the affinity peptide. Compared with the product manufactured according to the original process, these changes had no detectable effect on the structural integrity, stability or clinical efficacy of this antihaemophilia A product. The product produced by the improved manufacturing process is named XynthaÔ/ReFacto AF. [source] Nutrients Released by Gastric Epithelial Cells Enhance Helicobacter pylori GrowthHELICOBACTER, Issue 6 2004Karin Van Amsterdam ABSTRACT Background.,Helicobacter pylori survives and proliferates in the human gastric mucosa. In this niche, H. pylori adheres to the gastric epithelial cells near the tight junctions. In vitro, H. pylori proliferated well in tissue-culture medium near gastric epithelial cells. However, in the absence of epithelial cells, growth of H. pylori could only be established in tissue-culture medium when, prior to the experiment, it was preincubated near gastric epithelial cells. Therefore, we aimed to determine whether diffusion of nutrients derived from epithelial cells was required for H. pylori growth in Dulbecco's modified Eagle's minimal essential medium (DMEM) cell culture medium. Materials and Methods., Cell culture conditions essential for H. pylori growth in vitro were determined with gastric epithelial HM02 cells. Results., Deprivation of iron in cell-culture-conditioned DMEM resulted in a growth arrest of H. pylori. However, near gastric epithelial cells, growth of H. pylori was resistant to iron deprivation. Evidently, when residing close to epithelial cells, H. pylori was able to fulfil its iron requirements, even when the DMEM was deprived of iron. Nevertheless, supplementation with iron alone did not restore H. pylori growth in DMEM, hence other nutrients were deficient as well in the absence of epithelial cells. Growth of H. pylori in DMEM was restored when hypoxanthine, l -alanine and l -proline were added to the DMEM. Conclusions, Diffusion of (precursors of) these nutrients from the gastric epithelial cells is essential for H. pylori growth in vitro. We hypothesize that in vivo, H. pylori favors colonization near the tight junctions, to gain maximal access to the nutrient(s) released by gastric epithelial cells. [source] Cold-induced apoptosis of rat liver cells in University of Wisconsin solution: The central role of chelatable ironHEPATOLOGY, Issue 3 2002Uta Kerkweg Although University of Wisconsin (UW) solution aims at the prevention of cold-induced cell injury, it failed to protect against cold-induced apoptosis of hepatocytes and liver endothelial cells: when incubated in UW solution at 4°C for 24 hours and subsequently rewarmed at 37°C, 72% ± 8% of rat hepatocytes and 81% ± 5% of liver endothelial cells lost viability. In both cell types, the observed cell damage occurred under an apoptotic morphology; it appeared to be mediated by a rapid increase in the cellular chelatable iron pool by a factor ,2 (as determined in hepatocytes) and subsequent formation of reactive oxygen species (ROS). Consequently, this cell injury was decreased by iron chelators to 6 to 25% (hepatocytes) and 4% ± 2% (liver endothelial cells). Deferoxamine nearly completely inhibited the occurrence of apoptotic morphology in both cell types. In liver endothelial cells, cold-induced apoptosis occurring during rewarming after 24 hours of cold incubation in UW solution was far more pronounced than in cell culture medium (loss of viability: 81% ± 5% vs. 28% ± 13%), but viability could even be maintained for 2 weeks of cold incubation by use of deferoxamine. In conclusion, this pathological mechanism might be an explanation for the strong endothelial cell injury known to occur after cold preservation. With regard to the extent of this iron-mediated injury, addition of a suitable iron chelator to UW solution might markedly improve the outcome of liver preservation. [source] Antiproliferative activity of CCN3: Involvement of the C-terminal module and post-translational regulation,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2007A.M. Bleau Abstract Previous work had suggested that recombinant CCN3 was partially inhibiting cell proliferation. Here we show that native CCN3 protein secreted into the conditioned medium of glioma transfected cells indeed induces a reduction in cell proliferation. Large amounts of CCN3 are shown to accumulate both cytoplasmically and extracellularly as cells reach high density, therefore highlighting new aspects on how cell growth may be regulated by CCN proteins. Evidence is presented establishing that the amount of CCN3 secreted into cell culture medium is regulated by post-translational proteolysis. As a consequence, the production of CCN3 varies throughout the cell cycle and CCN3 accumulates at the G2/M transition of the cycle. We also show that CCN3-induced inhibition of cell growth can be partially reversed by specific antibodies raised against a C-terminal peptide of CCN3. The use of several clones expressing various portions of CCN3 established that the CT module of CCN3 is sufficient to induce cell growth inhibition. J. Cell. Biochem. 101: 1475,1491, 2007. © 2007 Wiley-Liss, Inc. [source] Nicotine inhibits myofibroblast differentiation in human gingival fibroblastsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2005Yiyu Fang Abstract Cigarette smoking has been suggested as a risk factor for several periodontal diseases. It has also been found that smokers respond less favorably than non-smokers to periodontal therapy. Previous work in our lab has shown that nicotine inhibits human gingival cell migration. Since myofibroblasts play an important role in wound closure, we asked if nicotine affects gingival wound healing process by regulating myofibroblast differentiation. Human gingival fibroblasts (HGFs) from two patients were cultured in 10% fetal bovine serum cell culture medium. Cells were pretreated with different doses of nicotine (0, 0.01, 0.1, and 1 mM) for 2 h, and then incubated with transforming growth factor beta (TGF-,1) (0, 0.25, 0.5, and 1 ng/ml) with or without nicotine for 30 h. The expression level of ,-smooth muscle actin (,-SMA), a specific marker for myofibroblasts, was analyzed by Western blots, immunocytochemistry, and real-time polymerase chain reaction (real-time PCR). Phosphorylated p38 mitogen-activated protein kinase (Phospho-p38 MAPK) activity was analyzed by Western blots. TGF-,1 induced an increase of ,-SMA protein and mRNA expression, while nicotine (1 mM) inhibited the TGF-,1-induced expression of ,-SMA but not ,-actin. Nicotine treatment down-regulated TGF-,1-induced p38 MAPK phosphorylation. Our results demonstrated for the first time that nicotine inhibits myofibroblast differentiation in human gingival fibroblasts in vitro; supporting the hypothesis that delayed wound healing in smokers may be due to decreased wound contraction by myofibroblasts. © 2005 Wiley-Liss, Inc. [source] Retinoic acid induces expression of the interleukin-1, gene in cultured normal human mammary epithelial cells and in human breast carcinoma linesJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2002Limin Liu Retinoic acid (RA) and its derivatives inhibit the proliferation of normal human mammary epithelial cells (HMEC) and some breast carcinoma lines by mechanisms which are not fully understood. To identify genes that mediate RA-induced cell growth arrest, an HMEC cDNA library was synthesized and subtractive screening was performed. We identified the interleukin-1, (IL-1,) gene as an RA induced gene in HMEC. Northern blot analyses showed that the IL-1, gene was up-regulated as early as 2 h after RA treatment. Results from the treatment of HMEC with cycloheximide and actinomycin D indicated that the regulation of the IL-1, gene by RA occurred at the transcriptional level and that the IL-1, gene is a direct, downstream target gene of RA. To evaluate the effects of IL-1, on cell proliferation, the proliferation of HMEC was measured in the presence of RA or IL-1,, or both. Either RA or IL-1, could significantly inhibit the proliferation of HMEC. However, the addition of soluble IL-1 receptor antagonist (sIL-1ra) to the cell culture medium did not block RA-induced HMEC growth inhibition, whereas sIL-1ra did block the growth inhibition of HMEC by IL-1,. IL-1, expression was not observed in the three carcinoma cell lines, MCF-7, MDA-MB-231, and MDA-MB-468, as compared to the HMEC. Growth curves of the breast carcinoma cell lines showed strong inhibitory effects of RA and IL-1, on the growth of the estrogen receptor (ER) positive MCF-7 cell line, but only a small effect on the ER negative MDA-MB-231 cells. The expression of the IL-1, gene was also transcriptionally activated by RA in normal epithelial cells of prostate and oral cavity. Our results suggest that: (a) the IL-1, gene is a primary target of RA receptors in HMEC; (b) the enhanced expression of the IL-1, gene does not mediate the RA-induced growth arrest of HMEC; and (c) the expression of the IL-1, gene is low or absent in all three human breast carcinoma cell lines examined, but the defect in the IL-1, signaling pathway may be different in ER positive versus ER negative carcinoma cells. © 2002 Wiley-Liss, Inc. [source] Extracorporeal photopheresis reduces the number of mononuclear cells that produce pro-inflammatory cytokines, when tested ex-vivoJOURNAL OF CLINICAL APHERESIS, Issue 4 2002John Bladon Abstract Extracorporeal photopheresis (ECP) has been shown to be clinically effective in the treatment of many T cell,mediated conditions. ECP's mechanism of action includes the induction of apoptosis and the release of pro-inflammatory cytokines. Recently, we have observed early lymphoid apoptosis, detectable immediately post ECP. We were interested to determine what influence ECP has on pro-inflammatory cytokine secretion at this early pre-infusion stage. Samples from 6 cutaneous T cell lymphoma (CTCL) and 5 graft versus host disease (GvHD) patients were taken pre ECP and immediately post ECP, prior to re-infusion. Following separation, the PBMCs were added to a cell culture medium and stimulated with PMA, Ionomycin, and Brefeldin A for 6 hours. Using flow cytometry, intracellular cytokine expression of IFN, and TNF, was determined in the T cell population. The monocytes were evaluated for IL6, IFN,, IL12, and TNF,. For both patient groups, the number of IFN,-expressing T cells fell significantly at re-infusion, whilst both T cell- and monocyte-expressing TNF, levels were reduced at re-infusion. All other cytokines tested showed no significant change post ECP. For GvHD, pro-inflammatory cytokines have a pathological role. Their down-regulation may have a direct clinical benefit. However, the reduction in the number of IFN,- and TNF,-expressing mononuclear cells means, at this early stage, it is unlikely that these cytokines assist in the removal of the malignant Th2 cells present in CTCL. J. Clin. Apheresis 17:177,182, 2002. © 2002 Wiley-Liss, Inc. [source] Cerebrotendinous xanthomatosis: molecular characterization of two Scandinavian sistersJOURNAL OF INTERNAL MEDICINE, Issue 3 2002E. Rystedt Abstract.,Rystedt E, Olin M, Seyama Y, Buchmann M, Berstad A, Eggertsen G, Björkhem I (Karolinska Institutet, Stockholm, Sweden; OchanomizuUniversity, Tokyo, Japan; Medisinsk avdeling, Haukeland sykehus, Bergen). Cerebrotendinous xanthomatosis: molecular characterization of two Scandinavian sisters (Case report). Journal of Internal Medicine 2002; 252: 259,264. Cerebrotendinous xanthomatosis (CTX) is a hereditary disorder, which is inherited as an autosomally recessive disease, causing production of cholesterol and cholestanol xanthomas and mental retardation. The disease is caused by mutations in the gene for sterol 27-hydroxylase (CYP27A1). The only CTX patients diagnosed in Scandinavia are two Norwegian sisters from a consanguineous marriage. Here we have characterized the mutation and its functional consequences for the enzyme. Analysis of genomic DNA from cultured fibroblasts identified a base exchange C > T in position 1441, causing arginine at amino acid position 441 to be replaced by tryptophan. The same mutation was introduced by mutagenesis in the complimentary DNA (cDNA) for CYP27, ligated into the expression vector pcDNA4/HisMax and transfected into HEK293 cells. The mutated enzyme had less than 5% of the enzyme activity compared with the native enzyme. No abnormal catalytic products could be identified in the cell culture medium. Probably the mutation affects the haem binding within the holoenzyme. The mutation has also previously been reported in a Japanese family. This is the second example of a CTX-causing mutation that has been recognized in more than one population. [source] Long-term cytotoxicity of resin-based dental restorative materialsJOURNAL OF ORAL REHABILITATION, Issue 1 2002S. Bouillaguet Highly filled composites, Ormocers (organically modified ceramics) and ,smart' materials have been developed to overcome the polymerization shrinkage problems of conventional composite materials. The purpose of the current study was to investigate the effect of longer-term (up to 8 weeks) ageing of these resin-based dental restorative materials and determine the effect of post-curing on cytotoxicity. Twelve discs of each material (Colombus/IDR, Definite/Degussa, Ariston pHc/Vivadent) were either light-cured (Lc) or light-cured and post-cured (Pc). For cytotoxicity testing, the discs were placed in contact with cell culture medium (DMEM) and incubated at 37 °C. Extracts from composite materials were collected after 24 h and weekly over a time period of 8 weeks. Cytotoxicity of the eluates to cultured fibroblasts (Balb/c3T3) were measured by the succinic dehydrogenase (SDH) activity (MTT assay) and the results expressed in percentage of negative controls (Teflon discs). The results showed that ageing significantly influenced the cytotoxicity of the materials. Except for Ariston pHc, materials were less cytotoxic after 8 weeks of ageing than they were in early intervals and post-curing was not generally useful in reducing cytotoxicity. The Ariston pHc was initially moderately toxic, but then become highly cytotoxic for 5 weeks before returning to initial levels. The current study demonstrated the importance of assessing the cytotoxicity of resin composite materials at multiple times. [source] Incorporation of proteins within alginate fibre-based scaffolds using a post-fabrication entrapment methodJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 7 2006Qingpu Hou In this study, a physical entrapment process was explored for the incorporation of proteins within preformed fibrous alginates and the release profile was tuned by varying the processing parameters. The entrapment process was carried out in a series of aqueous solutions at room temperature and involved pre-swelling of the fibrous alginate within a Na+ -rich solution, followed by exposure to the protein of choice and entrapping it by re-establishing cross-links of alginate with BaCl2. Entrapment and release of fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA), a model protein, was studied. It was found that a sustained release of the incorporated protein in cell culture medium for about 6 days was achieved. The main factors determining the release profile included the NaCl/CaCl2 ratio in the pre-swelling solution, protein concentration, and the exposure time. To retard protein release, alginate fibres with entrapped FITC-BSA were processed together with poly(d, l -lactide) (PDLLA) into porous alginate fibre/PDLLA composites using supercritical CO2. In this manner, release of the protein for up to 3 months was achieved. [source] EDTA enhances high-throughput two-dimensional bioprinting by inhibiting salt scaling and cell aggregation at the nozzle surfaceJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 4 2009Cheryl A. Parzel Abstract Tissue-engineering strategies may be employed in the development of in vitro breast tissue models for use in testing regimens of drug therapies and vaccines. The physical and chemical interactions that occur among cells and extracellular matrix components can also be elucidated with these models to gain an understanding of the progression of transformed epithelial cells into tumours and the ultimate metastases of tumour cells. The modified inkjet printer may be a useful tool for creating three-dimensional (3D) in vitro models, because it offers an inexpensive and high-throughput solution to microfabrication, and because the printer can be easily manipulated to produce varying tissue attributes. We hypothesized, however, that when ink is replaced with a biologically based fluid (i.e. a ,bio-ink'), specifically a serum-free cell culture medium, printer nozzle failure can result from salt scale build-up as fluid evaporates on the printhead surface. In this study, ethylene diamine tetra-acetic acid (EDTA) was used as a culture medium additive to prevent salt scaling and cell aggregation during the bioprinting process. The results showed that EDTA, at a concentration typically found in commercially available trypsin solutions (0.53 mM), prevented nozzle failure when a serum-free culture medium was printed from a nozzle at 1000 drops/s. Furthermore, increasing concentrations of EDTA appeared to mildly decrease aggregation of 4T07 cells. Cell viability studies were performed to demonstrate that addition of EDTA did not result in significant cell death. In conclusion, it is recommended that EDTA be incorporated into bio-ink solutions containing salts that could lead to nozzle failure. Copyright © 2009 John Wiley & Sons, Ltd. [source] The effect of the ionic products of Bioglass® dissolution on human osteoblasts growth cycle in vitroJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 4 2007Jun-Ying Sun Abstract In this study, in order to observe the effect of Bioglass® and its ionic products on human esteoblasts growth cycle in vitro, the ionic products of Bioglass have been introduced to a cell culture medium by dissolving Bioglass particles in Dulbecco's modified Eagle's medium (DMEM) at 37 °C for 24 h; this was used as the experimental medium, while DMEM without Bioglass modification was used as the control medium. Human osteoblasts isolated from trabecular bone were treated by the two media and the timing of the osteoblast growth cycle was examined. Cell growth curves were derived after 7 days. Also, human osteoblasts were treated for 1,6 days by the two media, and the G1, S, G2 phase percentages of osteoblasts were recorded by flow cytometry every day, resulting in the cell proliferation activity index: SPF (S-phase fraction) and PI (proliferation index). The difference in cell growth was shown after the second day of culture (p < 0.01), and cell growth in the experimental groups was greater than in control groups. The SPF and PI of the experimental groups were also higher than the control groups in 2 days of culture (p < 0.05 and p < 0.01), which indicates that the growth cycle of the human osteoblasts in experimental medium is about 2 days. In conclusion, Bioglass can promote osteoblast proliferation, reducing the human osteoblast growth cycle to pass through G1 and S phase and then enter G2 phase quickly. Copyright © 2007 John Wiley & Sons, Ltd. [source] Quantitative proteomics of intracellular Porphyromonas gingivalisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23 2007Qiangwei Xia Abstract Whole-cell quantitative proteomic analyses were conducted to investigate the change from an extracellular to intracellular lifestyle for Porphyromonas gingivalis, a Gram-negative intracellular pathogen associated with periodontal disease. Global protein abundance data for P. gingivalis strain ATCC 33277 internalized for 18,h within human gingival epithelial cells and controls exposed to gingival cell culture medium were obtained at sufficient coverage to provide strong evidence that these changes are profound. A total of 385 proteins were overexpressed in internalized P. gingivalis relative to controls; 240 proteins were shown to be underexpressed. This represented in total about 28% of the protein encoding ORFs annotated for this organism, and slightly less than half of the proteins that were observed experimentally. Production of several proteases, including the classical virulence factors RgpA, RgpB, and Kgp, was decreased. A separate validation study was carried out in which a 16-fold dilution of the P. gingivalis proteome was compared to the undiluted sample in order to assess the quantitative false negative rate (all ratios truly alternative). Truly null (no change) abundance ratios from technical replicates were used to assess the rate of quantitative false positives over the entire proteome. A global comparison between the direction of abundance change observed and previously published bioinformatic gene pair predictions for P. gingivalis will assist with future studies of P. gingivalis gene regulation and operon prediction. [source] Transport of Benzo[,]pyrene in the Dually Perfused Human Placenta Perfusion Model: Effect of Albumin in the Perfusion MediumBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 3 2009Line Mathiesen Foetal exposure to this substance is highly relevant but is difficult to estimate. The human placenta is unique compared to other species; since it is available without major ethical obstacles, we have used the human placenta perfusion model to study transport from mother to foetus. Placentas were donated after births at Rigshospitalet in Copenhagen from pregnant mothers who signed an informed consent. BaP is lipophilic and studies using cell culture medium in 6-hr placenta perfusions showed minimal transport through the placenta. To increase the solubility of BaP in perfusion medium and to increase physiological relevance, perfusions were also performed with albumin added to the perfusion medium [2 and 30 mg/ml bovine serum albumin (BSA) and 30 mg/ml human serum albumin (HSA)]. The addition of albumin resulted in increased transfer of BaP from maternal to foetal reservoirs. The transfer was even higher in the presence of an HSA formulation containing acetyltryptophanate and caprylate, resulting in a foetal,maternal concentration (FM) ratio of 0.71 ± 0.10 after 3 hr and 0.78 ± 0.11 after 6 hr, whereas the FM ratio in perfusions without albumin was only 0.05 ± 0.03 after 6 hr of perfusion. Less BaP accumulated in placental tissue in perfusions with added albumin. This shows that transplacental transport of the pro-carcinogenic substance BaP occurs, and emphasizes the importance of adding physiological concentrations of albumin when studying the transport of lipophilic substances. [source] Effects on Lipid Peroxidation and Antioxidative Enzymes of Euonymus alatus in Cultured Rat HepatocytesBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 1 2009Kyung-Woon Kim In this paper, we investigate the effects of E. alatus on cultured hepatocyte cell system and lipid peroxidation in hydrogen peroxide (H2O2) treatment conditions. The study covers the physiological activity (the antioxidative activity and the nitrite-scavenging effect) of E. alatus. H2O2 that can produce intracellular free radical was used for inducer of the peroxidation of cellular lipids. Treatment of E. alatus attenuated in cell killing enhanced by increasing concentrations of H2O2. The increased malondialdehyde level induced by H2O2 treatment was reduced by pre-treatment of E. alatus. Furthermore, addition of E. alatus in cell culture medium significantly reduced cell killing and content of intracellular antioxidants. Changes in nitrite-scavenging effect of E. alatus at various concentrations (5,25 mg/ml) and various pH levels (pH 1.2, 4.2 and 6.0) were also observed. The present study was also done to investigate the effects of E. alatus on cultured hepatocyte cell system, H2O2 -induced cytotoxicity and antioxidative enzyme activities, including catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferase in H2O2 treatment conditions. E. alatus treatment had significant protective or elevating activities on these antioxidative enzyme activities compared to a normal group. The results indicate that E. alatus provides a strong antioxidant protection of cells against H2O2 -induced oxidative stress. [source] Collapse temperature of solutions important for lyopreservation of living cells at ambient temperature,BIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Geer Yang Abstract In this study, the collapse temperature was determined using the freeze-drying microscopy (FDM) method for a variety of cell culture medium-based solutions (with 0.05,0.8,M trehalose) that are important for long-term stabilization of living cells in the dry state at ambient temperature (lyopreservation) by freeze-drying. Being consistent with what has been reported in the literature, the collapse temperature of binary water-trehalose solutions was found to be similar to the glass transition temperature (T,g , ,30°C) of the maximally freeze-concentrated trehalose solution (,80,wt% trehalose) during the freezing step of freeze-drying, regardless of the initial concentration of trehalose. However, the effect of the initial trehalose concentration on the collapse temperature of the cell culture medium-based trehalose solutions was identified to be much more significant, particularly when the trehalose concentration is less than 0.2,M (the collapse temperature can be as low as ,65°C). We also determined that cell density from 1 to 10,million cells/mL and ice seeding at high subzero temperatures (,4 and ,7°C) have negligible impact on the solution collapse temperature. However, ice seeding does significantly affect the ice crystal morphology formed during the freezing step and therefore the drying rate. Finally, bulking agents (mannitol) could significantly affect the collapse temperature only when trehalose concentration is low (<0.2,M). However, improving the collapse temperature by using a high concentration of trehalose might be preferred to the addition of bulking agents in the solutions for freeze-drying of living cells. We further confirmed the applicability of the collapse temperature measured with small-scale (2,µL) samples using the FDM system to freeze-drying of large-scale (1,mL) samples using scanning electron microscopy (SEM) data. Taken together, the results reported in this study should provide useful guidance to the development of optimal freeze-drying protocols for lyopreservation of living cells at ambient temperature for easy maintenance and convenient wide distribution to end users, which is important to the eventual success of modern cell-based medicine. Biotechnol. Bioeng. 2010;106: 247,259. © 2010 Wiley Periodicals, Inc. [source] Application of near-infrared (NIR) spectroscopy for screening of raw materials used in the cell culture medium for the production of a recombinant therapeutic proteinBIOTECHNOLOGY PROGRESS, Issue 2 2010Alime Ozlem Kirdar Abstract Control of raw materials based on an understanding of their impact on product attributes has been identified as a key aspect of developing a control strategy in the Quality by Design (QbD) paradigm. This article presents a case study involving use of a combined approach of Near-infrared (NIR) spectroscopy and Multivariate Data Analysis (MVDA) for screening of lots of basal medium powders based on their impact on process performance and product attributes. These lots had identical composition as per the supplier and were manufactured at different scales using an identical process. The NIR/MVDA analysis, combined with further investigation at the supplier site, concluded that grouping of medium components during the milling and blending process varied with the scale of production and media type. As a result, uniformity of blending, impurity levels, chemical compatibility, and/or heat sensitivity during the milling process for batches of large-scale media powder were deemed to be the source of variation as detected by NIR spectra. This variability in the raw materials was enough to cause unacceptably large variability in the performance of the cell culture step and impact the attributes of the resulting product. A combined NIR/MVDA approach made it possible to finger print the raw materials and distinguish between good and poor performing media lots. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Modeling kinetics of a large-scale fed-batch CHO cell culture by Markov chain Monte Carlo methodBIOTECHNOLOGY PROGRESS, Issue 1 2010Zizhuo Xing Abstract Markov chain Monte Carlo (MCMC) method was applied to model kinetics of a fed-batch Chinese hamster ovary cell culture process in 5,000-L bioreactors. The kinetic model consists of six differential equations, which describe dynamics of viable cell density and concentrations of glucose, glutamine, ammonia, lactate, and the antibody fusion protein B1 (B1). The kinetic model has 18 parameters, six of which were calculated from the cell culture data, whereas the other 12 were estimated from a training data set that comprised of seven cell culture runs using a MCMC method. The model was confirmed in two validation data sets that represented a perturbation of the cell culture condition. The agreement between the predicted and measured values of both validation data sets may indicate high reliability of the model estimates. The kinetic model uniquely incorporated the ammonia removal and the exponential function of B1 protein concentration. The model indicated that ammonia and lactate play critical roles in cell growth and that low concentrations of glucose (0.17 mM) and glutamine (0.09 mM) in the cell culture medium may help reduce ammonia and lactate production. The model demonstrated that 83% of the glucose consumed was used for cell maintenance during the late phase of the cell cultures, whereas the maintenance coefficient for glutamine was negligible. Finally, the kinetic model suggests that it is critical for B1 production to sustain a high number of viable cells. The MCMC methodology may be a useful tool for modeling kinetics of a fed-batch mammalian cell culture process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] A Cyclical Semicontinuous Process for Production of Human ,1 -Antitrypsin Using Metabolically Induced Plant Cell Suspension CulturesBIOTECHNOLOGY PROGRESS, Issue 2 2005Melody M. Trexler Transgenic rice suspension cultures were utilized to produce a human therapeutic protein, recombinant ,1 -antitrypsin (rAAT), in a cyclical, semicontinuous operation. Recombinant protein production was induced by removing the carbon source from the cell culture medium. The transgenic rice cells secreted the rAAT into the medium, and therefore medium exchanges could be performed for consecutive growth and protein expression phases. The process consisted of three cycles over a 25,28 day period, with growth phases lasting 4,6 days each and protein expression phases lasting 2.5,5 days each. Biomass and sugar concentrations, oxygen uptake rate, cell viability, culture pH, total extracellular protein, and active rAAT were measured throughout the cyclical process. The data profiles were reproducible between separate cyclical runs where, following each induction period, cell growth and viability could be reestablished once sucrose was added back to the culture. Volumetric productivities ranged from 3 to 12 mg active rAAT/(L day) for individual cycles with overall volumetric productivities of 4.5 and 7.7 mg active rAAT/(L day). [source] Parsing the Effects of Binding, Signaling, and Trafficking on the Mitogenic Potencies of Granulocyte Colony-Stimulating Factor AnaloguesBIOTECHNOLOGY PROGRESS, Issue 3 2003Casim A. Sarkar The pharmacodynamic potency of a therapeutic cytokine interacting with a cell-surface receptor can be attributed primarily to three central properties: [1] cytokine/receptor binding affinity, [2] cytokine/receptor endocytic trafficking dynamics, and [3] cytokine/receptor signaling. Thus, engineering novel or second-generation cytokines requires an understanding of the contribution of each of these to the overall cell response. We describe here an efficient method toward this goal in demonstrated application to the clinically important cytokine granulocyte colony-stimulating factor (GCSF) with a chemical analogue and a number of genetic mutants. Using a combination of simple receptor-binding and dose-response proliferation assays we construct an appropriately scaled plot of relative mitogenic potency versus ligand concentration normalized by binding affinity. Analysis of binding and proliferation data in this manner conveniently indicates which of the cytokine properties,binding, trafficking, and/or signaling,are contributing substantially to altered potency effects. For the GCSF analogues studied here, two point mutations as well as a poly(ethylene glycol) chemical conjugate were found to have increased potencies despite comparable or slightly lower affinities, and trafficking was predicted to be the responsible mechanism. A third point mutant exhibiting comparable binding affinity but reduced potency was predicted to have largely unchanged trafficking properties. Surprisingly, another mutant possessing an order-of-magnitude weaker binding affinity displayed enhanced potency, and increased ligand half-life was predicted to be responsible for this net beneficial effect. Each of these predictions was successfully demonstrated by subsequent measurements of depletion of these five analogues from cell culture medium. Thus, for the GCSF system we find that ligand trafficking dynamics can play a major role in regulating mitogenic potency. Our results demonstrate that cytokine analogues can exhibit pharmacodynamic behaviors across a diverse spectrum of "binding-potency space" and that our analysis through normalization can efficiently elucidate hypotheses for the underlying mechanisms for further dedicated testing. We have also extended the Black-Leff model of pharmacological agonism to include trafficking effects along with binding and signaling, and this model provides a framework for parsing the effects of these factors on pharmacodynamic potency. [source] N-[3,4-dimethoxycinnamoyl]-anthranilic acid (tranilast) suppresses microglial inducible nitric oxide synthase (iNOS) expression and activity induced by interferon-, (IFN-,)BRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2001Michael Platten Microglial cells up-regulate inducible nitric oxide synthase (iNOS) expression in response to various pro-inflammatory stimuli including interferon-, (IFN-,), allowing for the release of nitric oxide (NO). Tranilast (N-[3,4-dimethoxycinnamoyl]-anthranilic acid) is an antiallergic compound with suppressive effects on the activation of monocytes. Here, we show that N9 murine microglial cells express iNOS mRNA and protein and release nitric oxide into the culture medium in response to IFN-, (200 u ml,1) as measured by Northern and Western blot analyses and Griess assay. Exposure to non-toxic doses of tranilast (30 , 300 ,M) leads to a concentration-dependent inhibition of IFN-,-induced (200 u ml,1) iNOS mRNA and protein expression. This is paralleled by a suppression of NO-release into the cell culture medium. Inhibition of IFN-,-induced iNOS mRNA expression by tranilast is paralleled by an inhibition of nuclear factor-,B (NF-,B) activation and phosphorylation of inhibitory ,B (I,B) as determined by Western blot analyses and NF-,B reporter gene assay. These results suggest that tranilast-mediated suppression of microglial iNOS activity induced by IFN-, involves the inhibition of NF-,B-dependent iNOS mRNA expression. British Journal of Pharmacology (2001) 134, 1279,1284; doi:10.1038/sj.bjp.0704373 [source] Organ preservation solutions attenuate accumulation and nuclear translocation of hypoxia-inducible factor-1, in the hepatoma cell line HepG2CELL BIOCHEMISTRY AND FUNCTION, Issue 8 2009Renate Paddenberg Abstract Hypoxia-inducible factor-1, (HIF-1,) is a key transcription factor orchestrating hypoxic and inflammatory reactions. Here, we determined the impact of organ preservation solutions (Celsior; histidine-tryptophan-ketoglutarate solution, HTK; University of Wisconsin solution; UW), oxygen supply, and temperature on HIF-1, accumulation, recorded by Western blotting and immunocytochemistry, in the human hepatoma cell line HepG2. Generation of reactive oxygen species (ROS), NO, and cell viability were concomitantly assessed. At 4°C, HIF-1, accumulation was not detectable. In normothermic (37°C) cell culture medium (Dulbecco's Modified Eagle's Medium, DMEM), HepG2 cells accumulated HIF-1, even in normoxia (21% O2) which was not observed in either of the preservation solutions. This correlated to high generation of NO, a normoxic stabilizer of HIF-1,, and L -arginine content (substrate for NO synthesis) in DMEM, and low NO production and absence of L -arginine in preservation solutions. In normothermic hypoxia up to 24,h, intracellular HIF-1, accumulated in all conditions, but less in preservation solutions compared to DMEM. The inhibitory effect on accumulation and nuclear translocation was most prominent for HTK, the only solution containing the activator of HIF-1, degradation, , -ketoglutarate. Addition of other intermediates of the tricarbon acid cycle,succinate, fumarate, malate,did not alter HIF-1, accumulation, although succinate exhibited a beneficial effect on cell viability in cold storage. In conclusion, preservation solutions attenuate accumulation and nuclear translocation of the transcription factor HIF-1,, and this property is seemingly related to their chemical composition (L -arginine, , -ketoglutarate). Thus, it appears feasible to design preservation solution specifically to modify HIF-1, accumulation and nuclear translocation. Copyright © 2009 John Wiley & Sons, Ltd. [source] Activity and Stability of Hammerhead Ribozymes Containing 2,- C- Methyluridine: a New RNA MimicCHEMISTRY & BIODIVERSITY, Issue 2 2005Mariana Gallo We propose 2,- C -methylnucleotides as a new class of 2,-modified RNA mimics. These analogues are expected to provide 2,-OH groups capable of reproducing the interactions observed in natural RNA and, due to the presence of the Me group, to possess increased stability towards nucleases. In this work, we investigate the catalytic activity and nuclease resistance of hammerhead ribozymes carrying 2,- C -methyluridines in positions 4 and 7 of the catalytic core. We describe the in vitro activity of these chimeric molecules and their stability in cell lysate, fetal calf serum, and cell culture medium. The data show that, when only position 4 is modified, activity decreases twofold; while, when both 4 and 7 positions are substituted, a sevenfold drop in activity is observed. Regarding biological stability, the main increase of the half-life time is observed when position 7 is modified. These results suggest that 2,- C -methylnucleotides may be useful in the design of chemically synthesized RNA mimics with biological activity. [source] In vitro effects of cefotaxime and ceftriaxone on Salmonella typhi within human monocyte-derived macrophagesCLINICAL MICROBIOLOGY AND INFECTION, Issue 12 2002B. Ekinci The main objective of this in vitro study was to assess the effects of cefotaxime and ceftriaxone in killing Salmonella typhi in infected human macrophages. Human monocyte-derived macrophages isolated from peripheral blood of human volunteers were cultured in vitro for macrophage differentiation, and subsequently infected with S. typhi strains (a clinical isolate and a standard strain TA-42) at a cell ratio of 10 : 1. MICs of cefotaxime and ceftriaxone were determined by broth microdilution, and the antibiotics were included in the culture medium at one and five times their MIC values. Samples of cell culture medium taken at 0, 3, 6 and 24 h of incubation were cultured for growth of S. typhi on nutrient agar. Gentamicin (10 mg/L) was included in each well except for the control wells, in order to prevent growth of extracellular S. typhi. Both antibiotics showed good in vitro antibacterial effects against S. typhi strains. There were no statistically significant differences between the extracellular and intracellular effects of antibiotics with regard to elimination of the bacteria. Cefotaxime and ceftriaxone are highly effective against extracellular bacterial growth. The results of our in vitro experiments suggest that cefotaxime and ceftriaxone might also be used clinically against susceptible intracellular pathogens such as S. typhi. [source] Tissue engineering of periosteal cell membranes in vitroCLINICAL ORAL IMPLANTS RESEARCH, Issue 8 2009Patrick H. Warnke Abstract: Objectives: The cultivation of bone is a major focus in tissue engineering and oral implantology. Without a periosteal layer, instant or rapid development of a substantial cortical layer is unlikely for engineered bone grafts. The aim of this study was to test the ability of four collagen membranes to support and promote the proliferation of human periosteal cells. Materials and methods: Human periosteum cells were cultured using an osteogenic medium consisting of Dulbecco's modified Eagle's medium supplemented with fetal calf serum, penicillin, streptomycin and ascorbic acid at 37°C with 5% CO2. Four collagen membranes served as scaffolds: Bio-Gide, Chondro-Gide, Tutodent and Ossix Plus. Cell vitality was assessed by fluorescin diacetate (FDA) and propidium iodide (PI) staining, biocompatibility with LDH and BrdU, MTT, WST tests and scanning electron microscopy (SEM). Results: After 24 h, all probes showed viable periosteal cells. All biocompatibility tests revealed that proliferation on all membranes after treatment with eluate from membranes after a 24-h immersion in a serum-free cell culture medium was similar to the controls. Periosteal cells formed layers covering the surfaces of all four membranes 7 days after seeding in SEM. Conclusion: It can be concluded from our data that the collagen membranes can be used as scaffolds for the cultivation of periosteum layers with a view to creating cortical bone using tissue-engineering methods. [source] | |||||||||||||||||||||||||||||||