tetano
Editor, Senior Moderator
Vaccine. 2013 Oct 7. pii: S0264-410X(13)01314-5. doi: 10.1016/j.vaccine.2013.09.051. [Epub ahead of print]
Comparison of influenza virus yields and apoptosis-induction in an adherent and a suspension MDCK cell line.
Peschel B, Frentzel S, Laske T, Genzel Y, Reichl U.
Source
Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Sandtorstra?e 1, 39106 Magdeburg, Germany. Electronic address: britta.peschel@mpi-magdeburg.mpg.de.
Abstract
Cell culture-based manufacturing of influenza vaccines is ideally based on easily scalable platforms using suspension cells that grow in chemically defined media. Consequently, different adherent cell lines selected for high virus yields were adapted to grow in suspension culture. This includes the MDCK suspension cell line MDCK.SUS2, which was shown to be a suitable substrate for influenza virus propagation in previous studies. In this study, we investigated options for further improvement of influenza A/PR/8 (H1N1) virus titres and cell-specific virus yields. Best results were achieved by performing a 1:2 dilution with fresh medium at time of infection. In shake flask cultivations, even for multiplicities of infection as low as 10-5, all cells were infected at 36h post infection as determined by flow cytometry. In addition, these cells showed a better viability than cells infected without previous washing steps, which was reflected by a reduced level of apoptotic cells, and virus yields exceeding 3log10 HAU/100μL. Comparison of bioreactor infections of MDCK.SUS2 cells to the parental adherent MDCK cells showed similar HA titres of 2.94 and 3.15log10HAU/100μL and TCID50 of 1?109 and 2.37?109infectious virions/mL. Surprisingly, virus-induced apoptosis differed between the two cell lines, with the MDCK.SUS2 cells showing a much stronger apoptosis induction than the adherent MDCK cells. Obviously, despite their resistance to anoikis, the suspension cells were more susceptible to virus-induced apoptosis. Whether this is related to the adaptation process itself and/or to changes in cell survival pathways influenced by adhesion molecules or influenza virus proteins needs to be clarified in additional studies.
Copyright ? 2013. Published by Elsevier Ltd.
KEYWORDS:
1:2 dilution at toi, 1:2dil, 50% tissue culture infective dose, A/PR/8, A/Puerto Rico/8/34, CEACAM6, EMA, European medicines agency, FDA, FITC, Flow cytometry, Food and Drug Administration, GMEM, Glasgow minimum essential medium, HA, Influenza, MAD, MDCK, MDCK suspension cells, MOI, Madin?Darby canine kidney, Medium exchange, PI3K, TCID(50), TUNEL, TdT dUTP nick end labelling reaction, Trypsin activity, Vaccine, carcinoembryonic antigen-related cell adhesion molecule 6, fluorescein isothiocyanate, haemagglutinin, hours post infection, hpi, median of absolute deviation, multiplicity of infection, phosphatidylinositide 3-kinase, time of infection, toi, w/oME, wME, with medium exchange, without medium exchange
PMID:
24113260
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/24113260
Comparison of influenza virus yields and apoptosis-induction in an adherent and a suspension MDCK cell line.
Peschel B, Frentzel S, Laske T, Genzel Y, Reichl U.
Source
Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Sandtorstra?e 1, 39106 Magdeburg, Germany. Electronic address: britta.peschel@mpi-magdeburg.mpg.de.
Abstract
Cell culture-based manufacturing of influenza vaccines is ideally based on easily scalable platforms using suspension cells that grow in chemically defined media. Consequently, different adherent cell lines selected for high virus yields were adapted to grow in suspension culture. This includes the MDCK suspension cell line MDCK.SUS2, which was shown to be a suitable substrate for influenza virus propagation in previous studies. In this study, we investigated options for further improvement of influenza A/PR/8 (H1N1) virus titres and cell-specific virus yields. Best results were achieved by performing a 1:2 dilution with fresh medium at time of infection. In shake flask cultivations, even for multiplicities of infection as low as 10-5, all cells were infected at 36h post infection as determined by flow cytometry. In addition, these cells showed a better viability than cells infected without previous washing steps, which was reflected by a reduced level of apoptotic cells, and virus yields exceeding 3log10 HAU/100μL. Comparison of bioreactor infections of MDCK.SUS2 cells to the parental adherent MDCK cells showed similar HA titres of 2.94 and 3.15log10HAU/100μL and TCID50 of 1?109 and 2.37?109infectious virions/mL. Surprisingly, virus-induced apoptosis differed between the two cell lines, with the MDCK.SUS2 cells showing a much stronger apoptosis induction than the adherent MDCK cells. Obviously, despite their resistance to anoikis, the suspension cells were more susceptible to virus-induced apoptosis. Whether this is related to the adaptation process itself and/or to changes in cell survival pathways influenced by adhesion molecules or influenza virus proteins needs to be clarified in additional studies.
Copyright ? 2013. Published by Elsevier Ltd.
KEYWORDS:
1:2 dilution at toi, 1:2dil, 50% tissue culture infective dose, A/PR/8, A/Puerto Rico/8/34, CEACAM6, EMA, European medicines agency, FDA, FITC, Flow cytometry, Food and Drug Administration, GMEM, Glasgow minimum essential medium, HA, Influenza, MAD, MDCK, MDCK suspension cells, MOI, Madin?Darby canine kidney, Medium exchange, PI3K, TCID(50), TUNEL, TdT dUTP nick end labelling reaction, Trypsin activity, Vaccine, carcinoembryonic antigen-related cell adhesion molecule 6, fluorescein isothiocyanate, haemagglutinin, hours post infection, hpi, median of absolute deviation, multiplicity of infection, phosphatidylinositide 3-kinase, time of infection, toi, w/oME, wME, with medium exchange, without medium exchange
PMID:
24113260
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/24113260