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Animal Cell Cultures (animal + cell_culture)
Selected AbstractsPlant Protein Hydrolysates: Preparation of Defined Peptide Fractions Promoting Growth and Production in Animal Cells CulturesBIOTECHNOLOGY PROGRESS, Issue 5 2000Franti, ek Fran A new approach was applied with the aim at producing plant protein hydrolysates less heterogeneous and less contaminated with nonpeptide substances than are the presently available digests. A significant reduction of nonprotein contaminants was achieved by extraction of the plant material, soy flour or wheat flour, with acetone prior to isolation of the protein. Enzymes of nonanimal origin, papain or Pronase, were used for protein hydrolysis. The components of the hydrolysates were resolved by low-pressure liquid chromatography. Separation of peptide fractions and of remaining nonpeptide contaminants was achieved using small-pore size-exclusion chromatography matrices, Sephadex G-15 or Biogel P-2. Individual peptide fractions, both from soy protein and from wheat gluten, varied substantially in their growth-promoting and production-enhancing activities when tested on a mouse hybridoma culture in protein-free medium. The highest enhancement of viable cell density in batch cultures was 180% of control, and the highest enhancement of final immunoglobulin concentration was more than 230% of control. The existence of marked differences in activity of individual peptide fractions leads to a suggestion that the hydrolysates may provide peptides exerting specific positive effects on cultured animal cells. [source] Growth inhibition of dinoflagellate algae in shake flasks: Not due to shear this time!BIOTECHNOLOGY PROGRESS, Issue 1 2010Weiwei Hu Abstract Large scale algae cultures present interesting challenges in that they exhibit characteristics of typical bacterial and animal cell cultures. One current commercial food additive, docosahexaenoic acid (DHA), is produced using the dinoflagellate algae, Crypthecodiniumcohnii. Like animal cell culture, the perceived sensitivity of algae culture to hydrodynamic forces has potentially limited the agitation and aeration applied to these systems. However, the high density cultivation of C. cohnii required for an economically feasible process inevitably results in high oxygen demand. In this study, we demonstrated what first appeared to be a problem with shear sensitivity in shake flasks is most probably a mass transfer limitation. We subsequently demonstrated the limit of chronic and rapid energy dissipation rate, EDR, that C. cohnii cells can experience. This limit was determined using a microfluidic device connected in a recirculation loop to a stirred tank bioreactor, which has been previously used to repeatedly expose animal cells to high levels of EDR. Inhibition of cell growth was observed when C. cohnii cells were subjected to an EDR of 5.9 × 106 W/m3 with an average frequency of 0.2/min or more. This level of EDR is sufficiently high that C. cohnii can withstand typically encountered hydrodynamic forces in bioprocesses. This result suggests that at least one dinoflagellate algae, C. cohnii, is quite robust with respect to hydrodynamic forces and the scale-up of process using this type of algae should be more concerned with providing sufficient gas transfer given the relatively high oxygen demand. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Characterization of flow conditions in 2 L and 20 L wave bioreactors® using computational fluid dynamicsBIOTECHNOLOGY PROGRESS, Issue 1 2010Alper A. Öncül Abstract Characterization of flow conditions is of great importance to control cell growth and cell damage in animal cell culture because cell viability is influenced by the flow properties in bioreactors. Alternative reactor types like Wave Bioreactors® have been proposed in recent years, leading to markedly different results in cell growth and product formation. An advantage of Wave Bioreactors® is the disposability of the Polyethylenterephthalet-bags after one single use (fast setup of new production facilities). Another expected advantage is a lower shear stress compared to classical stirred-tank reactors, due to the gentle liquid motion in the rocking cellbag. This property would considerably reduce possible cell damage. The purpose of the present study is to investigate in a quantitative manner the key flow properties in Wave Bioreactors®, both numerically and experimentally. To describe accurately flow conditions and shear stress in Wave Bioreactors® using numerical simulations, it is necessary to compute the unsteady flow applying Computational Fluid Dynamics (CFD). Corresponding computations for two reactor scales (2 L and 20 L cellbags) are presented using the CFD code ANSYS-FLUENT®. To describe correctly the free liquid surface, the present simulations employ the Volume of Fluid (VOF) method. Additionally, experimental measurements have been carried out to determine liquid level, flow velocity and liquid shear stress, which are used as a validation of the present CFD simulations. It is shown that the obtained flows stay in the laminar regime. Furthermore, the obtained shear stress levels are well below known threshold values leading to damage of animal cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Inhibition of Human Cell Apoptosis by Silkworm HemolymphBIOTECHNOLOGY PROGRESS, Issue 4 2002Shin Sik Choi Many studies on preventing apoptosis have been carried out from the viewpoint of anti-apoptotic cloned-gene expressions inside cells, whereas in this study, we investigated the inhibition of apoptosis by the addition of silkworm hemolymph, a natural compound, from outside of the cells. In a previous study, we reported the inhibition effect of silkworm hemolymph on the baculovirus-induced insect cell apoptosis. Using the vaccinia virus-HeLa cell system as a model system in this study, we found that silkworm hemolymph, the insect serum, inhibits apoptosis not only in the insect cell system but also in the human cell system. The vaccinia virus-induced HeLa cell apoptosis was analyzed using DNA electrophoresis, TUNEL, and flow cytometry, and the resulting data confirmed that silkworm hemolymph inhibits human cell apoptosis. The inhibition of apoptosis due to silkworm hemolymph was not caused by an inhibition of virus binding and internalization steps, nor did silkworm hemolymph interfere with the virus production. The inhibition of apoptosis by silkworm hemolymph decreased the cell detachment from an adhering surface. With these characteristics, silkworm hemolymph can be effectively used to minimize cell death in commercial animal cell culture. [source] Approaches to achieve high-level heterologous protein production in plantsPLANT BIOTECHNOLOGY JOURNAL, Issue 1 2007Stephen J. Streatfield Summary Plants offer an alternative to microbial fermentation and animal cell cultures for the production of recombinant proteins. For protein pharmaceuticals, plant systems are inherently safer than native and even recombinant animal sources. In addition, post-translational modifications, such as glycosylation, which cannot be achieved with bacterial fermentation, can be accomplished using plants. The main advantage foreseen for plant systems is reduced production costs. Plants should have a particular advantage for proteins produced in bulk, such as industrial enzymes, for which product pricing is low. In addition, edible plant tissues are well suited to the expression of vaccine antigens and pharmaceuticals for oral delivery. Three approaches have been followed to express recombinant proteins in plants: expression from the plant nuclear genome; expression from the plastid genome; and expression from plant tissues carrying recombinant plant viral sequences. The most important factor in moving plant-produced heterologous proteins from developmental research to commercial products is to ensure competitive production costs, and the best way to achieve this is to boost expression. Thus, considerable research effort has been made to increase the amount of recombinant protein produced in plants. This research includes molecular technologies to increase replication, to boost transcription, to direct transcription in tissues suited for protein accumulation, to stabilize transcripts, to optimize translation, to target proteins to subcellular locations optimal for their accumulation, and to engineer proteins to stabilize them. Other methods include plant breeding to increase transgene copy number and to utilize germplasm suited to protein accumulation. Large-scale commercialization of plant-produced recombinant proteins will require a combination of these technologies. [source] NS0 cell damage by high gas velocity sparging in protein-free and cholesterol-free culturesBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2008Ying Zhu Abstract Recent developments in high cell density and high productivity fed-batch animal cell cultures have placed a high demand on oxygenation and carbon dioxide removal in bioreactors. The high oxygen demand is often met by increasing agitation and sparging rates of air/O2 in the bioreactors. However, as we demonstrate in this study, an increase of gas sparging can result in cell damage at the sparger site due to high gas entrance velocities. Previous studies have showed that gas bubble breakup at the culture surface was primarily responsible for cell damage in sparged bioreactors. Such cell damage can be reduced by use of surfactants such as Pluronic F-68 in the culture. In our results, where NS0 cells were grown in a protein-free and cholesterol-free medium containing 0.5 g/L Pluronic F-68, high gas entrance velocity at the sparger site was observed as the second mechanism for cell damage. Experiments were performed in scaled-down spinners to model the effect of hydrodynamic force resulting from high gas velocities on antibody-producing NS0 cells. Cell growth and cell death were described by first-order kinetics. Cell death rate constant increased significantly from 0.04 to 0.18 day,1 with increasing gas entrance velocity from 2.3 to 82.9 m/s at the sparger site. The critical gas entrance velocity for the NS0 cell line studied was found to be ,30 m/s; velocities greater than 30 m/s caused cell damage which resulted in reduced viability and consequently reduced antibody production. Observations from a second cholesterol-independent NS0 cell line confirmed the occurrence of cell damage due to high gas velocities. Increasing the concentration of Pluronic F-68 from 0.5 to 2 g/L had no additional protective effect on cell damage associated with high gas velocity at the sparger. The results of gas velocity analysis for cell damage have been applied in two case studies of large-scale antibody manufacturing. The first is a troubleshooting study for antibody production carried out in a 600 L bioreactor, and the second is the development of a gas sparger design for a large bioreactor scale (e.g., 10,000 L) for antibody manufacturing. Biotechnol. Bioeng. 2008;101: 751,760. © 2008 Wiley Periodicals, Inc. [source] Growth inhibition of dinoflagellate algae in shake flasks: Not due to shear this time!BIOTECHNOLOGY PROGRESS, Issue 1 2010Weiwei Hu Abstract Large scale algae cultures present interesting challenges in that they exhibit characteristics of typical bacterial and animal cell cultures. One current commercial food additive, docosahexaenoic acid (DHA), is produced using the dinoflagellate algae, Crypthecodiniumcohnii. Like animal cell culture, the perceived sensitivity of algae culture to hydrodynamic forces has potentially limited the agitation and aeration applied to these systems. However, the high density cultivation of C. cohnii required for an economically feasible process inevitably results in high oxygen demand. In this study, we demonstrated what first appeared to be a problem with shear sensitivity in shake flasks is most probably a mass transfer limitation. We subsequently demonstrated the limit of chronic and rapid energy dissipation rate, EDR, that C. cohnii cells can experience. This limit was determined using a microfluidic device connected in a recirculation loop to a stirred tank bioreactor, which has been previously used to repeatedly expose animal cells to high levels of EDR. Inhibition of cell growth was observed when C. cohnii cells were subjected to an EDR of 5.9 × 106 W/m3 with an average frequency of 0.2/min or more. This level of EDR is sufficiently high that C. cohnii can withstand typically encountered hydrodynamic forces in bioprocesses. This result suggests that at least one dinoflagellate algae, C. cohnii, is quite robust with respect to hydrodynamic forces and the scale-up of process using this type of algae should be more concerned with providing sufficient gas transfer given the relatively high oxygen demand. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Effects of Three-Dimensional Culturing on Osteosarcoma Cells Grown in a Fibrous Matrix: Analyses of Cell Morphology, Cell Cycle, and ApoptosisBIOTECHNOLOGY PROGRESS, Issue 5 2003Chunnuan Chen Osteosarcoma cells were cultured in stirred tank bioreactors with either a fibrous matrix or nonporous microcarriers to study the environmental effects on cell growth, morphology, cell cycle, and apoptosis. Cell cycle and apoptosis were analyzed using flow cytometry and visualized using confocal laser scanning microscopy and fluorescence microscopy. The three-dimensional (3-D) fibrous culture had better cell growth and higher metabolic rates than the two-dimensional (2-D) microcarrier culture because cells in the fibrous matrix were protected from shear stress and had lower apoptosis and cell death even under suboptimal conditions (e.g., nutrient depletion). The polyester fibrous matrix used in this study also exhibited the capability of selectively retaining viable and nonapoptotic cells and disposing apoptotic and nonviable cells. Consequently, very few apoptotic cells were found in the fibrous matrix even in the long-term (1 month) T-flask culture. In the continuous culture with packed fibrous matrixes for cell support, most cells were arrested in the G1/G0 phase after 4 days. Decreasing the dissolved oxygen level from 60 to 10% air saturation did not significantly change cell cycle and apoptosis, which remained low at ,15%. These results could explain why the fibrous bed bioreactor had good long-term stability and was advantageous for production of non-growth-associated proteins by animal cell cultures. [source] | ||||||||||