The Experts below are selected from a list of 237 Experts worldwide ranked by ideXlab platform
Udo Reichl - One of the best experts on this subject based on the ideXlab platform.
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Single-Cell Approach in Influenza Vaccine Production: Apoptosis and Virus Protein Production
Cells and Culture, 2010Co-Authors: J. Schulze-horsel, Yvonne Genzel, Mareike Schulze, Udo ReichlAbstract:The induction of apoptosis by influenza virus infection has been shown in vitro and in vivo. Here, we present a quantitative investigation of apoptosis occurring during influenza A vaccine production in Madin-Darby canine kidney Microcarrier Culture. Flow cytometry was employed for single-cell based analysis of infection, apoptosis and intracellular accumulation of viral nucleoprotein. Apoptotic DNA strand breaks were observed especially during late influenza A virus infection phase. Apoptosis was mainly detected in infected cells, only a small fraction of uninfected cells was apoptotic.
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infection dynamics and virus induced apoptosis in cell Culture based influenza vaccine production flow cytometry and mathematical modeling
Vaccine, 2009Co-Authors: J Schulzehorsel, Yvonne Genzel, Udo Reichl, M Schulze, G AgalaridisAbstract:Cell Culture-based influenza vaccine manufacturing is of growing importance. Depending on virus strains, differences in infection dynamics, virus-induced apoptosis, cell lysis and virus yields are observed. Comparatively little is known concerning details of virus-host cell interaction on a cellular level and virus spreading in a population of cells in bioreactors. In this study, the infection of MDCK cells with different influenza A virus strains in lab-scale Microcarrier Culture was investigated by flow cytometry. Together with the infection status of cells, virus-induced apoptosis was monitored. A mathematical model has been formulated to describe changes in the concentration of uninfected and infected adherent cells, dynamics of virus particle release (infectious virions, hemagglutinin content), and the time course of the percentage composition of the cell population.
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segregated mathematical model for growth of anchorage dependent mdck cells in Microcarrier Culture
Biotechnology Progress, 2008Co-Authors: L Mohler, A. Böck, Udo ReichlAbstract:To describe the growth behavior of anchorage-dependent mammalian cells in Microcarrier systems, various approaches comprising deterministic and stochastic single cell models as well as automaton-based models have been presented in the past. The growth restriction of these often contact-inhibited cells by spatial effects is described at levels with different complexity but for the most part not taking into account their metabolic background. Compared to suspension cell lines these cells have a comparatively long lag phase required for attachment and start of proliferation on the Microcarrier. After an initial phase of exponential growth only a moderate specific growth rate is achieved due to restrictions in space available for cell growth, limiting medium components, and accumulation of growth inhibitors. Here, a basic deterministic unstructured segregated cell model for growth of Madin Darby Canine Kidney (MDCK) cells used in influenza vaccine production is described. Four classes of cells are considered: cells on Microcarriers, cells in suspension, dead cells, and lysed cells. Based on experimental data, cell attachment and detachment is taken explicitly into account. The model allows simulation of the overall growth behavior in Microcarrier Culture, including the lag phase. In addition, it describes the time course of uptake and release of key metabolites and the identification of parameters relevant for the design and optimization of vaccine manufacturing processes.
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establishment of a mink enteritis vaccine production process in stirred tank reactor and wave bioreactor Microcarrier Culture in 1 10 l scale
Vaccine, 2007Co-Authors: Boris Hundt, N Schlawin, Holger Kassner, Yvonne Genzel, C. Best, Udo ReichlAbstract:Abstract A scale-up and process optimization scheme for the growth of adherent embryonic feline lung fibroblasts (E-FL) on Microcarriers and the propagation of a mink enteritis virus (MEV) strain for the production of an inactivated vaccine is shown. Stirred-tank cultivations are compared with results obtained from Wave® Bioreactors. Transfer from a roller bottle-based production process into large-scale Microcarrier Culture with starting concentrations of 2 g/L Cytodex™ 1 Microcarriers and 2.0 × 105 cells/mL was successful. A maximum cell yield of 1.2 × 106 cells/mL was obtained in stirred-tank Microcarrier batch Culture while cell numbers in the Wave® Bioreactor could not be determined accurately due to the fast sedimentation of Microcarriers under non-rocking conditions required for sampling. Detailed off-line analysis was carried out to understand the behaviour of the virus–host cell system in both cultivation systems. Metabolic profiles for glucose, lactate, glutamine, and ammonium showed slight differences for both systems. E-FL cell growth was on the same level in stirred-tank and Wave® Bioreactor with a higher volumetric cell yield compared to roller bottles. Propagation of MEV, which can only replicate efficiently in mitotic cells, was characterized in the Wave® Bioreactor using a multiple harvest strategy. Maximum virus titres of 106.6 to 106.8 TCID50/mL were obtained, which corresponds to an increase in virus yield by a factor of about 10 compared to cultivations in roller bottles. As a consequence, a single Wave® Bioreactor cultivation of appropriate scale can replace hundreds of roller bottles. Thus, the Wave® Bioreactor proved to be a suitable system for large-scale production of an inactivated MEV vaccine.
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mathematical model of influenza a virus production in large scale Microcarrier Culture
Biotechnology and Bioengineering, 2005Co-Authors: L Mohler, Udo Reichl, Heiner Sann, Dietrich FlockerziAbstract:A mathematical model that describes the replication of influenza A virus in animal cells in large-scale Microcarrier Culture is presented. The virus is produced in a two-step process, which begins with the growth of adherent Madin-Darby canine kidney (MDCK) cells. After several washing steps serum-free virus maintenance medium is added, and the cells are infected with equine influenza virus (A/Equi 2 (H3N8), Newmarket 1/93). A time-delayed model is considered that has three state variables: the number of uninfected cells, infected cells, and free virus particles. It is assumed that uninfected cells adsorb the virus added at the time of infection. The infection rate is proportional to the number of uninfected cells and free virions. Depending on multiplicity of infection (MOI), not necessarily all cells are infected by this first step leading to the production of free virions. Newly produced viruses can infect the remaining uninfected cells in a chain reaction. To follow the time course of virus replication, infected cells were stained with fluorescent antibodies. Quantitation of influenza viruses by a hemagglutination assay (HA) enabled the estimation of the total number of new virions produced, which is relevant for the production of inactivated influenza vaccines. It takes about 4-6 h before visibly infected cells can be identified on the Microcarriers followed by a strong increase in HA titers after 15-16 h in the medium. Maximum virus yield Vmax was about 1x10(10) virions/mL (2.4 log HA units/100 microL), which corresponds to a burst size ratio of about 18,755 virus particles produced per cell. The model tracks the time course of uninfected and infected cells as well as virus production. It suggests that small variations (<10%) in initial values and specific rates do not have a significant influence on Vmax. The main parameters relevant for the optimization of virus antigen yields are specific virus replication rate and specific cell death rate due to infection. Simulation studies indicate that a mathematical model that neglects the delay between virus infection and the release of new virions gives similar results with respect to overall virus dynamics compared with a time delayed model.
Qasim A. Rafiq - One of the best experts on this subject based on the ideXlab platform.
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Supplementary figures for "Process development of human multipotent stromal cell Microcarrier Culture using an automated high-throughput microbioreactor"
2018Co-Authors: Qasim A. Rafiq, Mariana P. Hanga, Thomas Heathman, Karen Coopman, Alvin Nienow, David Williams, Christopher HewittAbstract:Supplementary information files for "Process development of human multipotent stromal cell Microcarrier Culture using an automated high-throughput microbioreactor"Figure S1. Growth kinetics of hMSCs donor 2 cells using serum-free (SFM) and fetal bovine serum (FBS)-based media in both the ambr15 and spinner flasks with data showing the viable cell density.Figure S2. Nutrient and metabolite flux for hMSC donor 1 cells expanded on Microcarriers in the serum-based and serum-free Cultures in both the ambr and spinner flasks.Figure S3. Functional characterisation of hMSCs from donor 1 harvested from the serum-free ambr15 bioprocess.ABSTRACTMicrobioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and Culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell Culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) Microcarrier Culture processes. This was achieved by first improving suspension and mixing of the Microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in >250% increase in yield compared to the serum-based process. At both scales, the agitation used during Culture was the minimum required for Microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06–0.54%, respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC Microcarrier Cultures and that a combination of serum-free medium, control, and automation improves both process yield and consistency.
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process development of human multipotent stromal cell Microcarrier Culture using an automated high throughput microbioreactor
Biotechnology and Bioengineering, 2017Co-Authors: Qasim A. Rafiq, Mariana P. Hanga, Thomas Heathman, Karen Coopman, Alvin NienowAbstract:Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and Culture conditions. Here we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell Culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) Microcarrier Culture processes. This was achieved by first improving suspension and mixing of the Microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in > 150 % increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100 % working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in > 250% increase in yield compared to the serum-based process. At both scales, the agitation used during Culture was the minimum required for Microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06% and 0.54% respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC Microcarrier Cultures and that a combination of serum-free medium, control and automation improves both process yield and consistency.
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scalability and process transfer of mesenchymal stromal cell production from monolayer to Microcarrier Culture using human platelet lysate
Cytotherapy, 2016Co-Authors: Thomas Heathman, Qasim A. Rafiq, Karen Coopman, Alexandra Stolzing, Claire Fabian, A W NienowAbstract:Background aims: The selection of medium and associated reagents for human mesenchymal stromal cell (hMSC) Culture forms an integral part of manufacturing process development and must be suitable for multiple process scales and expansion technologies. Methods: In this work, we have expanded BM-hMSCs in fetal bovine serum (FBS)- and human platelet lysate (HPL)-containing media in both a monolayer and a suspension-based Microcarrier process. Results: The introduction of HPL into the monolayer process increased the BM-hMSC growth rate at the first experimental passage by 0.049 day and 0.127/day for the two BM-hMSC donors compared with the FBS-based monolayer process. This increase in growth rate in HPL-containing medium was associated with an increase in the inter-donor consistency, with an inter-donor range of 0.406 cumulative population doublings after 18 days compared with 2.013 in FBS-containing medium. Identity and quality characteristics of the BM-hMSCs are also comparable between conditions in terms of colony-forming potential, osteogenic potential and expression of key genes during monolayer and post-harvest from Microcarrier expansion. BM-hMSCs Cultured on Microcarriers in HPL-containing medium demonstrated a reduction in the initial lag phase for both BM-hMSC donors and an increased BM-hMSC yield after 6 days of Culture to 1.20 ± 0.17 × 105 and 1.02 ± 0.005 × 105 cells/mL compared with 0.79 ± 0.05 × 105 and 0.36 ± 0.04 × 105 cells/mL in FBS-containing medium. Conclusions: This study has demonstrated that HPL, compared with FBS-containing medium, delivers increased growth and comparability across two BM-hMSC donors between monolayer and Microcarrier Culture, which will have key implications for process transfer during scale-up.
Alvin Nienow - One of the best experts on this subject based on the ideXlab platform.
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Supplementary figures for "Process development of human multipotent stromal cell Microcarrier Culture using an automated high-throughput microbioreactor"
2018Co-Authors: Qasim A. Rafiq, Mariana P. Hanga, Thomas Heathman, Karen Coopman, Alvin Nienow, David Williams, Christopher HewittAbstract:Supplementary information files for "Process development of human multipotent stromal cell Microcarrier Culture using an automated high-throughput microbioreactor"Figure S1. Growth kinetics of hMSCs donor 2 cells using serum-free (SFM) and fetal bovine serum (FBS)-based media in both the ambr15 and spinner flasks with data showing the viable cell density.Figure S2. Nutrient and metabolite flux for hMSC donor 1 cells expanded on Microcarriers in the serum-based and serum-free Cultures in both the ambr and spinner flasks.Figure S3. Functional characterisation of hMSCs from donor 1 harvested from the serum-free ambr15 bioprocess.ABSTRACTMicrobioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and Culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell Culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) Microcarrier Culture processes. This was achieved by first improving suspension and mixing of the Microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in >250% increase in yield compared to the serum-based process. At both scales, the agitation used during Culture was the minimum required for Microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06–0.54%, respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC Microcarrier Cultures and that a combination of serum-free medium, control, and automation improves both process yield and consistency.
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process development of human multipotent stromal cell Microcarrier Culture using an automated high throughput microbioreactor
Biotechnology and Bioengineering, 2017Co-Authors: Qasim A. Rafiq, Mariana P. Hanga, Thomas Heathman, Karen Coopman, Alvin NienowAbstract:Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high-throughput screening of process parameters and Culture conditions. Here we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell Culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) Microcarrier Culture processes. This was achieved by first improving suspension and mixing of the Microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in > 150 % increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100 % working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum-based medium was applied to a serum-free process in the ambr15, resulting in > 250% increase in yield compared to the serum-based process. At both scales, the agitation used during Culture was the minimum required for Microcarrier suspension, NJS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06% and 0.54% respectively. The combination of both serum-free and automated processing improved the reproducibility more than 10-fold compared to the serum-based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC Microcarrier Cultures and that a combination of serum-free medium, control and automation improves both process yield and consistency.
J. M. Ossewaarde - One of the best experts on this subject based on the ideXlab platform.
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A Microcarrier Culture method for the production of large quantities of viable Chlamydia pneumoniae
Applied microbiology and biotechnology, 1996Co-Authors: A. Meijer, C. E. Vallinga, J. M. OssewaardeAbstract:We studied the propagation of Chlamydia pneumoniae strain TW-183 in HEp2 cells grown on Microcarrier beads. Infection of the cells in Microcarrier Culture was optimized by addition of 7.5% polyethylene glycol 4000 (PEG4000) during adsorption. The yield in Microcarrier Culture was similar to that of microtitre-plate Culture using centrifugation-assisted infection (120 x 10(6) and 225 x 10(6) bacteria/10(6) HEp2 cells respectively), as was the burst size (505 and 449 bacteria produced/infecting bacterium respectively). However, up to 64% savings in labour time and 27% saving in Culture medium were achieved if the Microcarrier Culture method was used instead of the microtitre-plate Culture method. The optimal yield of viable bacteria could only be achieved at a narrow range of multiplicities of infection (0.24 - 1.14 inclusion-forming units/cell), independent of the mode of infection (centrifugation-assisted infection of PEG4000-facilitated infection by adsorption) and independent of incubation temperature (35 degrees C or 37 degrees C). The yield of Microcarrier Cultures was the same at an incubation temperature of 35 degrees C or 37 degrees C in contrast to an increased production at 35 degrees C in the microtitre-plate Culture method using centrifugation-assisted infection. In conclusion, the Microcarrier Culture method is useful to produce large quantities of viable Chlamydia pneumoniae economically.
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A Microcarrier Culture method for the production of large quantities of viable Chlamydia pneumoniae
Applied microbiology and biotechnology, 1996Co-Authors: A. Meijer, C. E. Vallinga, J. M. OssewaardeAbstract:We studied the propagation of Chlamydia pneumoniae strain TW-183 in HEp2 cells grown on Microcarrier beads. Infection of the cells in Microcarrier Culture was optimized by addition of 7.5% polyethylene glycol 4000 (PEG4000) during adsorption. The yield in Microcarrier Culture was similar to that of microtitre-plate Culture using centrifugation-assisted infection (120×106 and 225×106 bacteria/106 HEp2 cells respectively), as was the burst size (505 and 449 bacteria produced/infecting bacterium respectively). However, up to 64% savings in labour time and 27% savings in Culture medium were achieved if the Microcarrier Culture method was used instead of the microtitre-plate Culture method. The optimal yield of viable bacteria could only be achieved at a narrow range of multiplicities of infection (0.24–1.14 inclusion-forming units/cell), independent of the mode of infection (centrifugation-assisted infection or PEG4000-facilitated infection by adsorption) and independent of incubation temperature (35°C or 37°C). The yield of Microcarrier Cultures was the same at an incubation temperature of 35°C or 37°C in contrast to an increased production at 35°C in the microtitre-plate Culture method using centrifugation-assisted infection. In conclusion, the Microcarrier Culture method is useful to produce large quantities of viable Chlamydia pneumoniae economically.
Yvonne Genzel - One of the best experts on this subject based on the ideXlab platform.
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Single-Cell Approach in Influenza Vaccine Production: Apoptosis and Virus Protein Production
Cells and Culture, 2010Co-Authors: J. Schulze-horsel, Yvonne Genzel, Mareike Schulze, Udo ReichlAbstract:The induction of apoptosis by influenza virus infection has been shown in vitro and in vivo. Here, we present a quantitative investigation of apoptosis occurring during influenza A vaccine production in Madin-Darby canine kidney Microcarrier Culture. Flow cytometry was employed for single-cell based analysis of infection, apoptosis and intracellular accumulation of viral nucleoprotein. Apoptotic DNA strand breaks were observed especially during late influenza A virus infection phase. Apoptosis was mainly detected in infected cells, only a small fraction of uninfected cells was apoptotic.
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infection dynamics and virus induced apoptosis in cell Culture based influenza vaccine production flow cytometry and mathematical modeling
Vaccine, 2009Co-Authors: J Schulzehorsel, Yvonne Genzel, Udo Reichl, M Schulze, G AgalaridisAbstract:Cell Culture-based influenza vaccine manufacturing is of growing importance. Depending on virus strains, differences in infection dynamics, virus-induced apoptosis, cell lysis and virus yields are observed. Comparatively little is known concerning details of virus-host cell interaction on a cellular level and virus spreading in a population of cells in bioreactors. In this study, the infection of MDCK cells with different influenza A virus strains in lab-scale Microcarrier Culture was investigated by flow cytometry. Together with the infection status of cells, virus-induced apoptosis was monitored. A mathematical model has been formulated to describe changes in the concentration of uninfected and infected adherent cells, dynamics of virus particle release (infectious virions, hemagglutinin content), and the time course of the percentage composition of the cell population.
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establishment of a mink enteritis vaccine production process in stirred tank reactor and wave bioreactor Microcarrier Culture in 1 10 l scale
Vaccine, 2007Co-Authors: Boris Hundt, N Schlawin, Holger Kassner, Yvonne Genzel, C. Best, Udo ReichlAbstract:Abstract A scale-up and process optimization scheme for the growth of adherent embryonic feline lung fibroblasts (E-FL) on Microcarriers and the propagation of a mink enteritis virus (MEV) strain for the production of an inactivated vaccine is shown. Stirred-tank cultivations are compared with results obtained from Wave® Bioreactors. Transfer from a roller bottle-based production process into large-scale Microcarrier Culture with starting concentrations of 2 g/L Cytodex™ 1 Microcarriers and 2.0 × 105 cells/mL was successful. A maximum cell yield of 1.2 × 106 cells/mL was obtained in stirred-tank Microcarrier batch Culture while cell numbers in the Wave® Bioreactor could not be determined accurately due to the fast sedimentation of Microcarriers under non-rocking conditions required for sampling. Detailed off-line analysis was carried out to understand the behaviour of the virus–host cell system in both cultivation systems. Metabolic profiles for glucose, lactate, glutamine, and ammonium showed slight differences for both systems. E-FL cell growth was on the same level in stirred-tank and Wave® Bioreactor with a higher volumetric cell yield compared to roller bottles. Propagation of MEV, which can only replicate efficiently in mitotic cells, was characterized in the Wave® Bioreactor using a multiple harvest strategy. Maximum virus titres of 106.6 to 106.8 TCID50/mL were obtained, which corresponds to an increase in virus yield by a factor of about 10 compared to cultivations in roller bottles. As a consequence, a single Wave® Bioreactor cultivation of appropriate scale can replace hundreds of roller bottles. Thus, the Wave® Bioreactor proved to be a suitable system for large-scale production of an inactivated MEV vaccine.
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metabolism of mdck cells during cell growth and influenza virus production in large scale Microcarrier Culture
Vaccine, 2004Co-Authors: Yvonne Genzel, Udo Reichl, Ilona Behrendt, Susanne Konig, Heiner SannAbstract:The production of equine influenza in Madin-Darby canine kidney (MDCK) cells in large-scale Microcarrier Culture is described with detailed on- and off-line analytical data during cell growth and virus replication. Metabolite concentration profiles for glucose, glutamine, lactate and ammonium are shown. Lactate and ammonium concentrations were always below inhibiting levels. Concentration profiles for essential and non-essential amino acids of the cell Culture medium are discussed. During cell growth proline was released into the medium with a significant rate while two amino acids, serine and methionine were almost depleted. After infection, virus titer increased after a delay of 10-16 h whereas first changes in amino acid metabolism could be observed within 4h post-infection. Here, glutamate and aspartate increase correlated to virus release kinetics, indicating cell disruption and apoptosis. Starting with a moi of 0.025 resulted in a maximum virus yield of 2.4 log HA/100 microl at 44 h post-infection.