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

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 ؋ 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.anchorage-independent ͉ hemagglutinin ͉ sialyltransferase ͉ vaccine

Monitoring changes in proteome during stepwise adaptation of a MDCK cell line from adherence to growth in suspension

“…For MDCK cells, an efficient host for influenza virus production [16,17], only few successful attempts to adapt cells to suspension growth have been reported [9,11,[18][19][20][21]. Of these only human siat7e gene expressing MDCK cells [11] and suspension cells derived over cell passage from adherent MDCK cells of the American and the European collection of cell cultures [9,21] are available for research purpose. As addressed above, such cell line adaptations always bear the risk to result in undesired cell line properties, and various adherent MDCK cell lines were repeatedly tested to be non-tumorigenic [7,20,22] while other MDCK cell variants were positively tested for tumorigenicity [7,20,23].…”

“…These include the use of optimized media such as SFM4BHK21 medium (HyClone) or EX-CELL ® medium (Sigma-Aldrich) [5,9], the application of dynamic cultivation systems like spinner flasks [10] and/or genetic modifications [11][12][13][14][15] as well as the generation of so-called designer cells with specific growth properties [3]. For MDCK cells, an efficient host for influenza virus production [16,17], only few successful attempts to adapt cells to suspension growth have been reported [9,11,[18][19][20][21]. Of these only human siat7e gene expressing MDCK cells [11] and suspension cells derived over cell passage from adherent MDCK cells of the American and the European collection of cell cultures [9,21] are available for research purpose.…”

Particle quantification of influenza viruses by high performance liquid chromatography

“…Furthermore, the World Health Organization (WHO) has established an action plan in 2006 to increase the current supply of influenza vaccine to 2 billion doses by 2015, which highlights the need to develop new technologies capable to support urgent and large demands for vaccines [6]. Therefore, several alternatives for rapid production have been developed or are being explored [7][8][9][10][11][12]. Some of these innovative production platforms only require 2-3 weeks to http://dx.doi.org/10.1016/j.vaccine.2014.…”

Rapid production of a H9N2 influenza vaccine from MDCK cells for protecting chicken against influenza virus infection

“…When an influenza virus is isolated in relation to an influenza pandemic, microcarrieranchored MDCK cells are an ideal choice to propagate the isolated influenza virus and to manufacture an inactivated influenza vaccine rapidly. As MDCK cells have been conversed to suspension culture (Chu et al 2009;van Wielink et al 2011), further work will be extended to perform investigations for the cultivation of different influenza virus strains in suspension MDCK cells.…”