41 research outputs found
Suspension culture of Pk15 cells for veterinary antigen production
The aim of this work is to characterize a culture of adherent pig kidney cells used for the production of an antigen for Porcine circoviral disease (PCVD), and determine a suitable strategy for scale up. The pig kidney cell line PK15 grown in adherence at low glucose concentration, was characterized in terms of culture and metabolic parameters, and subsequently adapted to suspension culture using a progressive adaptation method and media modification to a condition with low serum, high glucose and surfactant presence. The adapted cell was grown in suspension in 1 L flasks, and characterized also in terms of culture and metabolic parameters for comparison with the original adherent culture. Cells grown in suspension maintain a high viability and reach a similar maximum cell concentration as cells grown in adherence, while exhibiting a more efficient metabolic state. Cell concentration was increased by culture feeding, which is initiated at 5 mM residual glucose. Cells were infected with 103 viral genomic equivalents/cell, cultured and monitored for 120 hours. Cell growth and metabolic parameters were also determined. A stoichiometric model that considers the main metabolic pathways was used to assess and compare the metabolic state and needs of cells grown in adherence, grown in suspension and after infection using metabolic flux analysis. Results from this analysis were used to determine a suitable specific media composition for the cell growth stage and the viral infection stage in suspension culture. Results obtained in this study are to be transferred to the production setting.
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Validation of the model-based cell culture media design platform “CELIA” for biomass and product optimization in a bioreactor setting
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Mathematical modeling of the dynamic storage of iron in ferritin
<p>Abstract</p> <p>Background</p> <p>Iron is essential for the maintenance of basic cellular processes. In the regulation of its cellular levels, ferritin acts as the main intracellular iron storage protein. In this work we present a mathematical model for the dynamics of iron storage in ferritin during the process of intestinal iron absorption. A set of differential equations were established considering kinetic expressions for the main reactions and mass balances for ferritin, iron and a discrete population of ferritin species defined by their respective iron content.</p> <p>Results</p> <p>Simulation results showing the evolution of ferritin iron content following a pulse of iron were compared with experimental data for ferritin iron distribution obtained with purified ferritin incubated <it>in vitro </it>with different iron levels. Distinctive features observed experimentally were successfully captured by the model, namely the distribution pattern of iron into ferritin protein nanocages with different iron content and the role of ferritin as a controller of the cytosolic labile iron pool (cLIP). Ferritin stabilizes the cLIP for a wide range of total intracellular iron concentrations, but the model predicts an exponential increment of the cLIP at an iron content > 2,500 Fe/ferritin protein cage, when the storage capacity of ferritin is exceeded.</p> <p>Conclusions</p> <p>The results presented support the role of ferritin as an iron buffer in a cellular system. Moreover, the model predicts desirable characteristics for a buffer protein such as effective removal of excess iron, which keeps intracellular cLIP levels approximately constant even when large perturbations are introduced, and a freely available source of iron under iron starvation. In addition, the simulated dynamics of the iron removal process are extremely fast, with ferritin acting as a first defense against dangerous iron fluctuations and providing the time required by the cell to activate slower transcriptional regulation mechanisms and adapt to iron stress conditions. In summary, the model captures the complexity of the iron-ferritin equilibrium, and can be used for further theoretical exploration of the role of ferritin in the regulation of intracellular labile iron levels and, in particular, as a relevant regulator of transepithelial iron transport during the process of intestinal iron absorption.</p
Parameters for cell growth, ΔL/ΔH<sup>a</sup>, IgG’s specific productivity and their percentage variation vs. their respective control cultures.
<p><sup>a</sup>H stands for hexose, depending on the culture’s conditions it can be either glucose (G) or fructose (F).</p><p>Parameters for cell growth, ΔL/ΔH<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119053#t001fn001" target="_blank"><sup>a</sup></a>, IgG’s specific productivity and their percentage variation vs. their respective control cultures.</p
Comparative Metabolic Analysis of CHO Cell Clones Obtained through Cell Engineering, for IgG Productivity, Growth and Cell Longevity
<div><p>Cell engineering has been used to improve animal cells’ central carbon metabolism. Due to the central carbon metabolism’s inefficiency and limiting input of carbons into the TCA cycle, key reactions belonging to these pathways have been targeted to improve cultures’ performance. Previous works have shown the positive effects of overexpressing PYC2, MDH II and fructose transporter. Since each of these modifications was performed in different cell lines and culture conditions, no comparisons between these modifications can be made. In this work we aim at contrasting the effect of each of the modifications by comparing pools of transfected IgG producing CHO cells cultivated in batch cultures. Results of the culture performance of engineered clones indicate that even though all studied clones had a more efficient metabolism, not all of them showed the expected improvement on cell proliferation and/or specific productivity. CHO cells overexpressing PYC2 were able to improve their exponential growth rate but IgG synthesis was decreased, MDH II overexpression lead to a reduction in cell growth and protein production, and cells transfected with the fructose transporter gene were able to increase cell density and reach the same volumetric protein production as parental CHO cells in glucose. We propose that a redox unbalance caused by the new metabolic flux distribution could affect IgG assembly and protein secretion. In addition to reaction dynamics, thermodynamic aspects of metabolism are also discussed to further understand the effect of these modifications over central carbon metabolism.</p></div