140,261 research outputs found
Limits and benefits of the interpretation of extracellular metabolite data in mammalian cell culture
No full textBefore considering intracellular metabolites clear ideas on the extracellular conditions of experiments are obligatory. Many interesting questions on understanding metabolism of cells are generated under process conditions and not under defined steady state conditions with defined media.
Here, we discuss limits and benefits of the interpretation of extracellular metabolite data for a process of influenza vaccine production in mammalian cells. Therefore, the production process is introduced together with relevant details on medium composition and cultivation conditions. Typical issues of metabolism during cell growth and viral infection are presented. Data on basic metabolites (glucose, lactate, glutamine, ammonia) are shown together with amino acid profiles measured from the cell culture medium [1]. Comparing different media (serum-containing, glutamine-free, serum-free [2-4]) as well as different cultivation methods (roller bottles, stirred tank bioreactor, wave bioreactor) it is demonstrated, what can be learned from extracellular metabolites and what questions remain.
[1] Genzel Y.; König S.; Reichl U.; "Amino acid analysis in mammalian cell culture media containing serum and high glucose concentrations by anion exchange chromatography and integrated pulsed amperometric detection (IPAD)." , Anal. Biochem. 2004, 335, 119-125.
[2] Genzel Y.; Behrendt I.; König S.; Sann H.; Reichl U.; "Metabolism of MDCK cells during cell growth and influence virus production in large-scale microcarrier culture" , Vaccine 2004, 22(17-18), 2202-2208.
[3] Genzel Y.; Ritter JB.; König S.; Alt R.; Reichl U.; "Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells." , Biotechnol. Progr. 2005, 21 (1), 58-69.
[4] Genzel Y.; Fischer M.; Reichl U.; "Serum-free influenza virus production avoiding washing steps and medium exchange in large-scale microcarrier culture.", Vaccine 2006, 24(16), 3261-3272
Limits and benefits of the interpretation of extracellular metabolite data in mammalian cell culture
No full textBefore considering intracellular metabolites clear ideas on the extracellular conditions of experiments are obligatory. Many interesting questions on understanding metabolism of cells are generated under process conditions and not under defined steady state conditions with defined media. Here, we discuss limits and benefits of the interpretation of extracellular metabolite data for a process of influenza vaccine production in mammalian cells. Therefore, the production process is introduced together with relevant details on medium composition and cultivation conditions. Typical issues of metabolism during cell growth and viral infection are presented. Data on basic metabolites (glucose, lactate, glutamine, ammonia) are shown together with amino acid profiles measured from the cell culture medium [1]. Comparing different media (serum-containing, glutamine-free, serum-free [2-4]) as well as different cultivation methods (roller bottles, stirred tank bioreactor, wave bioreactor) it is demonstrated, what can be learned from extracellular metabolites and what questions remain. [1] Genzel Y.; König S.; Reichl U.; "Amino acid analysis in mammalian cell culture media containing serum and high glucose concentrations by anion exchange chromatography and integrated pulsed amperometric detection (IPAD)." , Anal. Biochem. 2004, 335, 119-125. [2] Genzel Y.; Behrendt I.; König S.; Sann H.; Reichl U.; "Metabolism of MDCK cells during cell growth and influence virus production in large-scale microcarrier culture" , Vaccine 2004, 22(17-18), 2202-2208. [3] Genzel Y.; Ritter JB.; König S.; Alt R.; Reichl U.; "Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells." , Biotechnol. Progr. 2005, 21 (1), 58-69. [4] Genzel Y.; Fischer M.; Reichl U.; "Serum-free influenza virus production avoiding washing steps and medium exchange in large-scale microcarrier culture.", Vaccine 2006, 24(16), 3261-3272
Participación ciudadana, organizaciones de la sociedad civil y fortalecimiento institucional del Tribunal de Cuentas de la Provincia de Jujuy
No full textTesis de MaestríaSe expone la participación ciudadana y su potencial aporte a la generación de valor público a través de una guía de acción que contribuya a fortalecer la rendición de cuentas del Tribunal de Cuentas de la Provincia de Jujuy, mediante la interacción de instituciones intermedias que tributen a un mecanismo de intervención que permita monitorear, de manera institucionalizada, las actividades de control fomentando la cultura de la transparencia y facilitando un canal de diálogo con la Entidad de Fiscalización Superior. El objetivo es proponer un modelo de acercamiento cooperativo de actores con los cuales interactuar en base a capacidades propias y competencias profesionales, generando un espacio de comunicación directa que habilite un trabajo complementario, redunde en elevar los niveles de confianza y legitimidad del Tribunal de Cuentas y, a la vez, evidencie el valor y beneficio que generará en la vida de los ciudadanos.Fil: Genzel, Marcelo Germán. Universidad Nacional de San Martín. Escuela de Política y Gobiern
Changes in specific enzyme activities of MDCK cells during growth under different cultivation conditions
No full textVarious producer cell lines used for large scale production of biopharmaceuticals like monoclonal antibodies, hormones or viral vaccines often show inefficient use of nutrients for growth. They have a very high demand in substrates, mainly glucose and glutamine, which results in release of large amounts of lactate and ammonium. This metabolic imbalance often not only decreases cell productivity and viability but also inhibits growth to high cell densities. Thus, new strategies including optimization of cultivation conditions, design of media and modification of specific properties of cells by systems biology approaches should be developed to increase cell densities and process yields.
For the growth of adherent MDCK cells distinct differences in glycolysis and tricarboxylic acid cycle were found for extra- and intracellular metabolite concentrations and metabolic fluxes when comparing the influence of glutamine or pyruvate addition to cell culture media [1-4].
The objective of this study was to further elucidate these differences by establishing a high-throughput platform for enzyme activity measurements of mammalian cells [5]. This method, which uses four cycling assays, allowed the determination of 28 key enzyme activities of central carbon metabolism in extracts of MDCK cells. Whereas, a comparison of Pyr and Gln-containing medium showed an up-regulation of glutamine synthetase activity by a factor of 4, the activity of glutaminase was down-regulated by a factor of 6 in GMEM medium. Decreased activities were also found for ATP-citrate lyase, phosphoenolpyruvate carboxykinase and the glutaminolytic enzymes aspartate- and alanine transaminase with Pyr as carbon source. Under all conditions, very low activities of pyruvate dehydrogenase and pyruvate carboxylase were measured and confirmed by comparing normal mouse kidney tissue with MDCK cells. Other enzymes like lactate dehydrogenase (LDH) and malate dehydrogenase showed less distinct changes in activity. In all media the activities of the glycolytic enzymes hexokinase and phosphofructokinase were relatively low, whereas maximum activities of LDH and pyruvate kinase were comparatively high. In contrast, most enzyme activities measured in serum-free Episerf medium were increased.
Based on the established assay metabolic states of production cell lines can now be further characterized. This can then be used to validate mathematical models of cellular metabolism and to improve our understanding of intracellular metabolic interactions relevant for process characterization and optimization.
[1] Genzel, Y., Ritter, J.B., König, S., Alt, R. and Reichl, U. 2005. Biotechnol. Prog., 21, 58-69.
[2] Ritter, J.B., Genzel, Y. and Reichl, U. 2006. Journal of Chromatography B, 843, 216-226.
[3] Sidorenko, Y., Wahl, A., Dauner, M., Genzel, Y. and Reichl, U. 2008. Biotechnol Prog, 24(2), 311-320.
[4] Wahl, A., Sidorenko, Y., Dauner, M., Genzel, Y. & Reichl, U. 2008. Biotechnology and Bioengineering, 101(1), 135-152.
[5] Janke, R., Genzel, Y., Wahl, A & Reichl, U. 2010. Metabolic Engineering, submitted
Changes in specific enzyme activities of MDCK cells during growth under different cultivation conditions
No full textVarious producer cell lines used for large scale production of biopharmaceuticals like monoclonal antibodies, hormones or viral vaccines often show inefficient use of nutrients for growth. They have a very high demand in substrates, mainly glucose and glutamine, which results in release of large amounts of lactate and ammonium. This metabolic imbalance often not only decreases cell productivity and viability but also inhibits growth to high cell densities. Thus, new strategies including optimization of cultivation conditions, design of media and modification of specific properties of cells by systems biology approaches should be developed to increase cell densities and process yields. For the growth of adherent MDCK cells distinct differences in glycolysis and tricarboxylic acid cycle were found for extra- and intracellular metabolite concentrations and metabolic fluxes when comparing the influence of glutamine or pyruvate addition to cell culture media [1-4]. The objective of this study was to further elucidate these differences by establishing a high-throughput platform for enzyme activity measurements of mammalian cells [5]. This method, which uses four cycling assays, allowed the determination of 28 key enzyme activities of central carbon metabolism in extracts of MDCK cells. Whereas, a comparison of Pyr and Gln-containing medium showed an up-regulation of glutamine synthetase activity by a factor of 4, the activity of glutaminase was down-regulated by a factor of 6 in GMEM medium. Decreased activities were also found for ATP-citrate lyase, phosphoenolpyruvate carboxykinase and the glutaminolytic enzymes aspartate- and alanine transaminase with Pyr as carbon source. Under all conditions, very low activities of pyruvate dehydrogenase and pyruvate carboxylase were measured and confirmed by comparing normal mouse kidney tissue with MDCK cells. Other enzymes like lactate dehydrogenase (LDH) and malate dehydrogenase showed less distinct changes in activity. In all media the activities of the glycolytic enzymes hexokinase and phosphofructokinase were relatively low, whereas maximum activities of LDH and pyruvate kinase were comparatively high. In contrast, most enzyme activities measured in serum-free Episerf medium were increased. Based on the established assay metabolic states of production cell lines can now be further characterized. This can then be used to validate mathematical models of cellular metabolism and to improve our understanding of intracellular metabolic interactions relevant for process characterization and optimization. [1] Genzel, Y., Ritter, J.B., König, S., Alt, R. and Reichl, U. 2005. Biotechnol. Prog., 21, 58-69. [2] Ritter, J.B., Genzel, Y. and Reichl, U. 2006. Journal of Chromatography B, 843, 216-226. [3] Sidorenko, Y., Wahl, A., Dauner, M., Genzel, Y. and Reichl, U. 2008. Biotechnol Prog, 24(2), 311-320. [4] Wahl, A., Sidorenko, Y., Dauner, M., Genzel, Y. & Reichl, U. 2008. Biotechnology and Bioengineering, 101(1), 135-152. [5] Janke, R., Genzel, Y., Wahl, A & Reichl, U. 2010. Metabolic Engineering, submitted
Effects of different growth conditions on the catalytic activities of central metabolic enzymes in MDCK cells
No full textIt is of common knowledge that Madin-Darby canine kidney (MDCK) cells are very suitable for the propagation of different influenza strains and, therefore, for the production of cell culture-based vaccines [1]. However, during growth in glutamine-containing media, the glycolytic and glutaminolytic fluxes of most cell lines are up-regulated and large amounts of toxic by-products, such as lactate and ammonia, are secreted into the medium. This metabolic imbalance often not only affects cell viability and productivity but also can prevent growth to high cell densities [2,3]. A promising approach to reduce waste-products is the substitution of one or several components in the culture medium [4,5]. In glutamine-free medium with pyruvate as carbon source, MDCK cells not only released no ammonia during cell growth but glucose consumption and lactate production was also reduced significantly [4]. In previous work with MDCK cells, several assays were developed, to determine the extra- and intracellular metabolite concentrations [6]. Furthermore, mathematical models were established to analyze the switch from glutamine-containing to glutamine-free (pyruvate) medium [7]. However, concerning the interpretation of experimental data and corresponding flux distributions, still some open questions remain. The objective of this study was to further elucidate the impact of media changes on metabolism by establishing a high-throughput platform for enzyme activity measurements of mammalian cells [8]. The method established uses four sensitive enzymatic cycling assays, and allows the determination of 28 key enzyme activities of central carbon metabolism in extracts of MDCK cells. Adherent MDCK cells were grown to stationary and exponential phases in 6-well plates in serum-containing GMEM supplemented with glutamine or pyruvate as well as in serum-free EPISERF medium, and key metabolic enzyme activities of cell extracts were analyzed. Significant differences were found in maximal enzyme activities from cells grown with pyruvate-containing medium compared to glutamine-containing medium. In particular, the overall activity of the pentose phosphate pathway was up-regulated during exponential cell growth in pyruvate-containing medium, which suggests that more glucose 6-phosphate was channeled into the oxidative branch and therefore more NADPH was required. Furthermore, the anaplerotic enzymes pyruvate carboxylase and pyruvate dehydrogenase showed higher cell specific activities with pyruvate, indicating an increased flux into the TCA cycle. An increase was also found for NAD+-dependent isocitrate dehydrogenase, glutamate dehydrogenase and glutamine synthetase, which is a strong indicator for an increased flux through the right part of the citrate cycle in MDCK cells grown with pyruvate. It can be assumed that extracellular pyruvate was directly shunted into the TCA cycle, and that the increase in enzyme activities was most likely required to compensate for the energy demand and to replenish the glutamine pool. On the other hand, the activities of the glutaminolytic enzymes aspartate transaminase, alanine transaminase, malic enzyme and phosphoenolpyruvate carboxykinase were decreased in cells grown with pyruvate, which seems to be related to a decreased glutamine metabolism. Based on the established enzyme assays metabolic states of production cell lines can now be further characterized. This can then be used to validate mathematical models of cellular metabolism and to improve our understanding of intracellular metabolic interactions relevant for process characterization and optimization. [1] Genzel, Y., Reichl, U., (2009). Expert Rev Vaccines 8, 1681-1692. [2] Glacken, M.W., (1988). Bio-Technol 6, 1041-1050. [3] Ozturk, S.S., Riley, M.R., Palsson, B.O., (1992). Biotechnol and Bioeng 39, 418-431. [4] Genzel, Y., Ritter, J.B., König, S., Alt, R., Reichl, U., (2005). Biotechnol Prog 21, 58-69. [5] Butler, M., Christie, A., (1994). Cytotechnology 15, 87-94. [6] Ritter, J.B., Genzel, Y., Reichl, U., (2008). Anal Biochem 373, 349-369. [7] Sidorenko, Y., Wahl, A., Dauner, M., Genzel, Y., Reichl, U., (2008). Biotechnol Progr 24, 311-320. [8] Janke, R., Genzel, Y., Wahl, A., Reichl, U., (2010). Biotechnol and Bioeng 107, 566-581
Effects of different growth conditions on the catalytic activities of central metabolic enzymes in MDCK cells
No full textIt is of common knowledge that Madin-Darby canine kidney (MDCK) cells are very suitable for the propagation of different influenza strains and, therefore, for the production of cell culture-based vaccines [1]. However, during growth in glutamine-containing media, the glycolytic and glutaminolytic fluxes of most cell lines are up-regulated and large amounts of toxic by-products, such as lactate and ammonia, are secreted into the medium. This metabolic imbalance often not only affects cell viability and productivity but also can prevent growth to high cell densities [2,3]. A promising approach to reduce waste-products is the substitution of one or several components in the culture medium [4,5]. In glutamine-free medium with pyruvate as carbon source, MDCK cells not only released no ammonia during cell growth but glucose consumption and lactate production was also reduced significantly [4]. In previous work with MDCK cells, several assays were developed, to determine the extra- and intracellular metabolite concentrations [6]. Furthermore, mathematical models were established to analyze the switch from glutamine-containing to glutamine-free (pyruvate) medium [7]. However, concerning the interpretation of experimental data and corresponding flux distributions, still some open questions remain.
The objective of this study was to further elucidate the impact of media changes on metabolism by establishing a high-throughput platform for enzyme activity measurements of mammalian cells [8]. The method established uses four sensitive enzymatic cycling assays, and allows the determination of 28 key enzyme activities of central carbon metabolism in extracts of MDCK cells. Adherent MDCK cells were grown to stationary and exponential phases in 6-well plates in serum-containing GMEM supplemented with glutamine or pyruvate as well as in serum-free EPISERF medium, and key metabolic enzyme activities of cell extracts were analyzed.
Significant differences were found in maximal enzyme activities from cells grown with pyruvate-containing medium compared to glutamine-containing medium. In particular, the overall activity of the pentose phosphate pathway was up-regulated during exponential cell growth in pyruvate-containing medium, which suggests that more glucose 6-phosphate was channeled into the oxidative branch and therefore more NADPH was required. Furthermore, the anaplerotic enzymes pyruvate carboxylase and pyruvate dehydrogenase showed higher cell specific activities with pyruvate, indicating an increased flux into the TCA cycle. An increase was also found for NAD+-dependent isocitrate dehydrogenase, glutamate dehydrogenase and glutamine synthetase, which is a strong indicator for an increased flux through the right part of the citrate cycle in MDCK cells grown with pyruvate. It can be assumed that extracellular pyruvate was directly shunted into the TCA cycle, and that the increase in enzyme activities was most likely required to compensate for the energy demand and to replenish the glutamine pool. On the other hand, the activities of the glutaminolytic enzymes aspartate transaminase, alanine transaminase, malic enzyme and phosphoenolpyruvate carboxykinase were decreased in cells grown with pyruvate, which seems to be related to a decreased glutamine metabolism.
Based on the established enzyme assays metabolic states of production cell lines can now be further characterized. This can then be used to validate mathematical models of cellular metabolism and to improve our understanding of intracellular metabolic interactions relevant for process characterization and optimization.
[1] Genzel, Y., Reichl, U., (2009). Expert Rev Vaccines 8, 1681-1692.
[2] Glacken, M.W., (1988). Bio-Technol 6, 1041-1050.
[3] Ozturk, S.S., Riley, M.R., Palsson, B.O., (1992). Biotechnol and Bioeng 39, 418-431.
[4] Genzel, Y., Ritter, J.B., König, S., Alt, R., Reichl, U., (2005). Biotechnol Prog 21, 58-69.
[5] Butler, M., Christie, A., (1994). Cytotechnology 15, 87-94.
[6] Ritter, J.B., Genzel, Y., Reichl, U., (2008). Anal Biochem 373, 349-369.
[7] Sidorenko, Y., Wahl, A., Dauner, M., Genzel, Y., Reichl, U., (2008). Biotechnol Progr 24, 311-320.
[8] Janke, R., Genzel, Y., Wahl, A., Reichl, U., (2010). Biotechnol and Bioeng 107, 566-581
El rol del taller de cocina como un acontecimiento que posibilita la multiplicación de las identificaciones
No full textPráctica Supervisada (Licenciatura en Psicología) -- Universidad Nacional de Córdoba. Facultad de Psicología, 2025.Fil: Genzel, Agustina. Universidad Nacional de Córdoba. Facultad de Psicología; Argentina.Fil: Lasa, Matías Joel. Universidad Nacional de Córdoba. Facultad de Psicología; Argentina.El siguiente trabajo fue realizado con el propósito de plasmar lo vivenciado en los siete meses de Prácticas Supervisadas transcurridas dentro de la Fundación Azud, las cuales son una modalidad de egreso de la Facultad de Psicología de la Universidad Nacional de Córdoba. En este marco, se realiza un acercamiento al rol profesional. Se apunta al análisis y reflexión, a partir de una articulación teórico-práctica, sobre el rol del taller de cocina como un acontecimiento que permite la multiplicación de las identificaciones. Se tuvieron en consideración aportes de la perspectiva vincular del Psicoanálisis en busca de una lectura que nos permitiera reconstruir nuestra experiencia haciendo énfasis en lo observado durante los encuentros que se generaban en los talleres de cocina. Se considera finalmente la importancia de las interacciones que se despliegan en los talleres de cocina del Centro de Día y, a partir de ello, la posibilidad de pensar modos de transitar la cotidianeidad de manera dinámica, en permanente transformación y donde la relación con otros/as posibilita que lo novedoso acontezca.Fil: Genzel, Agustina. Universidad Nacional de Córdoba. Facultad de Psicología; Argentina.Fil: Lasa, Matías Joel. Universidad Nacional de Córdoba. Facultad de Psicología; Argentina
Perfusion system in high-density cell culture for higher yields in vaccine production
No full textProduct yields of biologicals such as monoclonal antibodies, recombinant proteins and vaccines produced in mammalian cell culture have to be improved constantly to cope with increasing demands and process economics. As an example we investigate an influenza vaccine production process with adherently growing MDCK cells [Genzel et al., 2004]. The process consists of a cell growth phase and a virus replication phase. We want to achieve high-density cell cultures and finally higher virus yields in microcarrier systems. Main focus is on batch-to-batch reproducibility and scale-up. To quantitatively analyze this process various mathematical models are being developed to describe phenomena such as attachment, proliferation and contact inhibition of cells. In the past, we achieved higher cell numbers in small scale bioreactors using different control strategies for perfusion systems during cell growth [Bock et al., 2005]. A perfusion mode with cell retention by filtration allowed a continuous medium exchange preventing substrate limitations, and metabolite inhibitions. So far, the virus titers (HA) did not increase in the same magnitude as expected from cell numbers. Similar results were obtained by Genzel et al, 2005 in an influenza vaccine process in a wave® bioreactor system and by Pohlscheidt et al., 2005 in a parapoxvirus vaccine production process. Furthermore, Pohlscheidt et al. demonstrated a successful increase in virus titers by using a 20 kDa dialysis system or an Expanded-Volume-Batch during virus replication. Therefore, a perfusion system might also be successfully applied during influenza virus replication. In addition, the removal of virus particles from the culture broth might increase the virus yield (HA) by avoiding unspecific degradation of virions by enzymes released from lysed cells. Here, we present results concerning the influence of perfusion rates during virus replication on virus yields. Bock et al., 2005. Closed loop control of perfusion systems in high-density cell culture. Proceedings of the 19th meeting of ESACT in Harrogate, United Kingdom Genzel et al., 2005. Serum-free influenza production with MDCK cells in wave-bioreactor and 5L-stirred tank bioreactor. Proceedings of the 19th meeting of ESACT in Harrogate, United Kingdom Pohlscheidt et al., 2005. Strategies for large scale production of Parapoxvirus Ovis by micro-carrier cell culture. Proceedings of the 19th meeting of ESACT in Harrogate, United Kingdo
Perfusion system in high-density cell culture for higher yields in vaccine production
No full textProduct yields of biologicals such as monoclonal antibodies, recombinant proteins and vaccines produced in mammalian cell culture have to be improved constantly to cope with increasing demands and process economics. As an example we investigate an influenza vaccine production process with adherently growing MDCK cells [Genzel et al., 2004]. The process consists of a cell growth phase and a virus replication phase. We want to achieve high-density cell cultures and finally higher virus yields in microcarrier systems. Main focus is on batch-to-batch reproducibility and scale-up. To quantitatively analyze this process various mathematical models are being developed to describe phenomena such as attachment, proliferation and contact inhibition of cells.
In the past, we achieved higher cell numbers in small scale bioreactors using different control strategies for perfusion systems during cell growth [Bock et al., 2005]. A perfusion mode with cell retention by filtration allowed a continuous medium exchange preventing substrate limitations, and metabolite inhibitions.
So far, the virus titers (HA) did not increase in the same magnitude as expected from cell numbers. Similar results were obtained by Genzel et al, 2005 in an influenza vaccine process in a wave® bioreactor system and by Pohlscheidt et al., 2005 in a parapoxvirus vaccine production process. Furthermore, Pohlscheidt et al. demonstrated a successful increase in virus titers by using a 20 kDa dialysis system or an Expanded-Volume-Batch during virus replication. Therefore, a perfusion system might also be successfully applied during influenza virus replication. In addition, the removal of virus particles from the culture broth might increase the virus yield (HA) by avoiding unspecific degradation of virions by enzymes released from lysed cells. Here, we present results concerning the influence of perfusion rates during virus replication on virus yields.
Bock et al., 2005. Closed loop control of perfusion systems in high-density cell culture. Proceedings of the 19th meeting of ESACT in Harrogate, United Kingdom
Genzel et al., 2005. Serum-free influenza production with MDCK cells in wave-bioreactor and 5L-stirred tank bioreactor. Proceedings of the 19th meeting of ESACT in Harrogate, United Kingdom
Pohlscheidt et al., 2005. Strategies for large scale production of Parapoxvirus Ovis by micro-carrier cell culture. Proceedings of the 19th meeting of ESACT in Harrogate, United Kingdo
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