Is the „The Lord of the Rings“-Trilogy the ideal way to produce movies?A Description of the Developmentprocess of the Trilogy in comparison to the Productionsections of a Blockbuster Production

nicht vorhande

Molecular Targets for Gastric Cancer Treatment and Future Perspectives from a Clinical and Translational Point of View

Gastric cancer is a leading cause of cancer death worldwide. Systemic treatment comprising chemotherapy and targeted therapy is the standard of care in advanced/metastatic gastric cancer. Comprehensive molecular characterization of gastric adenocarcinomas by the TCGA Consortium and ACRG has resulted in the definition of distinct molecular subtypes. These efforts have in parallel built a basis for the development of novel molecularly stratified treatment approaches. Based on this molecular characterization, an increasing number of specific genomic alterations can potentially serve as treatment targets. Consequently, the development of promising compounds is ongoing. In this review, key molecular alterations in gastric and gastroesophageal junction cancers will be addressed. Finally, the current status of the translation of targeted therapy towards clinical applications will be reviewed

Multiple glycerol shocks increase the calcium phosphate transfection of non-synchronized CHO cells

The exposure of CHO DG44 cells to an osmotic shock, after DNA uptake, results in a cellular volume decrease of approx. 55%. Repetitive osmotic shocks targeted different sub-populations of cells as was demonstrated using two different fluorescent reporter genes. Also the exposure of a calcium phosphate-DNA coprecipitate to high osmolarity in vitro caused the release of the DNA from the precipitate. The results demonstrate the importance of the osmotic shock on the efficient delivery of plasmid DNA to the nucleus of CHO cells following calcium phosphate-mediated transfectio

Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives

The expansion of the biologics pipeline depends on the identification of candidate proteins for clinical trials. Speed is one of the critical issues, and the rapid production of high quality, research-grade material for preclinical studies by transient gene expression (TGE) is addressing this factor in an impressive way: following DNA transfection, the production phase for TGE is usually 2-10days. Recombinant proteins (r-proteins) produced by TGE can therefore enter the drug development and screening process in a very short time--weeks. With "classical” approaches to protein expression from mammalian cells, it takes months to establish a productive host cell line. This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studie

The Kinetics of Polyethylenimine-Mediated Transfection in Suspension Cultures of Chinese Hamster Ovary Cells

The kinetics of polyethylenimine (PEI)-mediated gene transfer at early times after transfection of Chinese hamster ovary (CHO) cell in suspension were investigated using a novel in vitro assay. Addition of an excess of competitor DNA to the culture medium at various times after the initiation of transfection inhibited further cellular uptake of PEI-DNA particles. Using this approach, a constant rate of particle uptake was observed during the first 60min of transfection at a PEI:DNA ratio of 2:1 (w/w) and a cell density of 2×106cells/ml under serum-free conditions. The uptake rate declined considerably during the next 2h of transfection. Both the rate and the level of PEI-DNA uptake in serum-free minimal medium were found to be dependent on the PEI-DNA ratio, the cell density at the time of transfection, and the extent of particle aggregation. These studies of the early phase of PEI-mediated transfection are expected to lead to further opportunities for optimization of gene transfer to suspension cultures of mammalian cells for the purpose of large-scale transient recombinant protein productio

Reduced glutamine concentration improves protein production in growth-arrested CHO-DG44 and HEK-293E cells

For most cultivated mammalian cells, glutamine is an essential medium component. However, glutamine consumption results in the production of ammonia, a cytotoxic byproduct. Here we investigated the effect of glutamine reduction on recombinant protein production and ammonia accumulation in transiently transfected CHO and HEK-293E cells maintained under conditions of growth arrest. Maximum transient recombinant protein yields were observed in HEK-293E cultures without glutamine and in CHO cultures with 2mM glutamine. The initial concentration of glutamine correlated with the level of ammonia accumulation in each culture. For both a stable CHO-derived cell line and a polyclonal population of recombinant CHO cells grown under conditions of mild hypothermia, the highest volumetric protein productivity was observed in cultures without glutamine. Here, the level of ammonia accumulation also corresponded to the initial glutamine concentration. Our data demonstrate that reduction of glutamine in the medium is an effective approach to improve protein production in both transiently and stably transfected mammalian cells when applying conditions that reduce or arrest the growth of these cell

Hyperosmolarity enhances transient recombinant protein yield in Chinese hamster ovary cells

The effect of hyperosmolarity on transient recombinant protein production in Chinese hamster ovary (CHO) cells was investigated. Addition of 90mM NaCl to the production medium ProCHO5 increased the volumetric yield of recombinant antibody up to 4-fold relative to transfection in ProCHO5 alone. Volumetric yields up to 50mgl−1 were achieved in a 6day batch culture of 3l. In addition, hyperosmolarity reduced cell growth and increased cell size. The addition of salt to cultures of transiently transfected CHO cells is a simple and cost-effective method to increase TGE yields in this hos

Study of the improved Sf9 transient gene expression process

Insect cells have been widely used for the production of recombinant proteins using recombinant baculovirus for gene delivery [1]. To simplify protein production in insect cells, we have previously described a method, based on transient gene expression (TGE) with cultures of suspension-adapted Sf9 cells using polyethylenimine (PEI) for DNA delivery [2]. Expression of GFP has been realized at high efficiency and a tumor necrosis factor receptor-Fc fusion protein (TNFR-Fc) was produced at a level of 40 mg/L. However, the efficiency of the insect cells TGE system has not been studied and further opti- mization may improve protein titers. Here, we studied the efficiency of PEI for plasmid delivery in Sf9 cells

Surfactant doped polyaniline coatings for functionalized gas diffusion layers in low temperature fuel cells

Gas diffusion layers (GDLs) are essential for the proper distribution of the reaction gases, the removal of excess water as well as electrical contact in polymer electrolyte fuel cells (PEFCs). The production of state-of-the-art GDLs consists of many steps such as graphitization at high temperatures and hydrophobic treatments with polytetrafluoroethylene (PTFE) which increase the cost. In this study, an electrically conductive and hydrophobic polyaniline (PANI) coating was deposited on carbon paper via dip-coating and electropolymerization to fabricate PTFE-free GDLs. As a proof-of-concept, PANI-coated GDLs were tested as a cathodic GDL in a single cell PEFC and achieved a 42% higher maximum power compared to the reference measurement with a commercial GDL. Furthermore, these PTFE-free GDLs achieved contact angles up to 144° which is in the range of commercial GDLs. The chemical composition of the PANI-coating was investigated via infrared spectroscopy and energy dispersive X-ray spectroscopy (EDX) and the morphology was examined via scanning electron microscopy (SEM). Hence, the proposed method emerges as a possible strategy to simultaneously substitute PTFE and apply a protective and durable coating.</p

TubeSpin bioreactor 50 for the high-density cultivation of Sf-9 insect cells in suspension

Here we present the TubeSpin bioreactor 50 (TubeSpins) as a simple and disposable culture system for Sf-9 insect cells in suspension. Sf-9 cells had substantially better growth in TubeSpins than in spinner flasks. After inoculation with 106cells/ml, maximal cell densities of 16×106 and 6×106cells/ml were reached in TubeSpins and spinner flasks, respectively. In addition the cell viability in these batch cultures remained above 90% for 10days in TubeSpins but only for 4days in spinner flasks. Inoculation at even higher cell densities reduced the duration of the lag phase. After inoculation at 2.5×106cells/ml, the culture reached the maximum cell density within 3days instead of 7days as observed for inoculation with 106cells/ml. Infection of Sf-9 cells in TubeSpins or spinner flasks with a recombinant baculovirus coding for green fluorescent protein (GFP) resulted in similar GFP-specific fluorescence levels. TubeSpins are thus an attractive option for the small-scale cultivation of Sf-9 cells in suspension and for baculovirus-mediated recombinant protein productio