Conversion of MDCK cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production

Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 09/2009; 106(35):14802-7. DOI: 10.1073/pnas.0905912106
Source: PubMed

ABSTRACT MDCK cells are currently being considered as an alternative to embryonated eggs for influenza virus propagation and hemagglutinin (HA) production intended for vaccine manufacturing. MDCK cells were found suitable for the virus production but their inability to grow in suspension burdens the process of scale up and hence their production capability. Anchorage-dependent MDCK cells were converted to anchorage-independent cells, capable of growing in suspension as a result of transfection with the human siat7e gene (ST6GalNac V). This gene was previously identified as having an important role in cellular adhesion when the transcriptions of genes from anchorage-dependent and anchorage-independent HeLa cells were compared. Unlike the parental MDCK cells, the siat7e-expressing cells were capable of growing in shake flasks as suspension cultures, achieving maximum concentration of 7 x 10(5) cells/mL while keeping close to 100% viability throughout the growth phase. In production experiments, the siat7e-expressing cells were infected with the Influenza B/Victoria/504/2000 strain. It was determined that the cell-derived viruses retained similar antigenic properties as those obtained from egg-derived viruses and their nucleotide sequences were identical. The specific production of hemagglutinin (expressed in hemagglutination units per 10(6) cells) from the siat7e-expressing cells was approximately 20 times higher than the specific production from the parental MDCK cells. If this suspension process scales up, the production potential of HA from 10 L of siat7e-expressing cells at a concentration of 10(6) cells/mL would be equivalent to the amount of HA obtained from 10,000 embryonated eggs.

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    • "Alternatively, some cellular components may inhibit host protein synthesis during transfection. Targeted engineering of the MDCK and Hela cell lines in order to increase the capability to replicate influenza virus has been described previously (Chu et al., 2009; Furuse et al., 2009; Hossain et al., 2010; Stech et al., 2008; Yang et al., 2010). This may open ways to achieve better transfection and expression efficacy. "
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    • "Influenza pandemics remain a serious public health problem worldwide. Influenza virus-associated illnesses cause an estimated 200,000–500,000 hospital admissions and hundreds of thousands of deaths annually (Simonsen et al., 2000; Suwannakarn et al., 2010; Cox and Subbarao, 2000; Webby and Webster, 2003; Wang et al., 2010; Chu et al., 2009). The emergence of variations in virus strains is one of the principal challenges facing prevention of infection with influenza virus. "
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    • "Many different approaches are available to develop high expressing cell lines for production of influenza virus. For example, the adaptation of adherent cells to grow in suspension cultures with the help of microcarriers or bioinformatics and genetic engineering technologies is a valuable tool for industrial applications due to easy scale-up and high cell density (Chu et al. 2009; Edwards et al. 2009). MDCK-SIAT1 cells which were engineered to express increased level of a-2, 6-linked sialic acid receptor were found to be superior to conventional MDCK cells for isolation of human influenza virus (Oh et al. 2008). "
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