Conversion of MDCK cell line to suspension culture
by transfecting with human siat7e gene and its
application for influenza virus production
Chia Chua,b, Vladimir Lugovtsevc, Hana Goldingc, Michael Betenbaugha, and Joseph Shiloachb,1
aDepartment of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218; andbBiotechnology Core
Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, andcCenter for Biologics Evaluation and
Research, Food and Drug Administration, 9000 Rockville Pike, Bethesda, MD 20892
Communicated by John B. Robbins, National Institutes of Health, Bethesda, MD, July 14, 2009 (received for review March 10, 2009)
MDCK cells are currently being considered as an alternative to
embryonated eggs for influenza virus propagation and hemagglu-
tinin (HA) production intended for vaccine manufacturing. MDCK
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 grow-
ing 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 ? 105
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 pro-
duction of hemagglutinin (expressed in hemagglutination units
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 106
cells/mL would be equivalent to the amount of HA obtained from
10,000 embryonated eggs.
anchorage-independent ? hemagglutinin ? sialyltransferase ? vaccine
annually (1). In response to rapid antigenic drift in influenza
viruses, the most effective approach taken has been the distri-
bution of trivalent inactivated viral vaccines, which are tradi-
tionally produced in chicken embryonated eggs (2). However, in
the event of a pandemic outbreak, this egg-based production
system may not be adequate to meet the surge in demand quickly
enough. The limitations associated with egg-based vaccines,
which include reliable egg supplies, prolonged cultivation peri-
ods, and cumbersome operations have spurred exploration of
alternatives. Among the potential alternatives for vaccine pro-
duction, the use of characterized, immortalized cell lines (par-
ticularly MDCK, VERO, and PER.C6) has been investigated.
These cell lines have been found to produce consistently high
viral titers (3–8). Nevertheless, one of the limiting aspects in
scaling up the virus production in these continuous cell lines is
the fact that these cells are anchorage-dependent and thus
require surface adhesion to proliferate (9, 10). Without surface
attachment, these cells cannot exert their normal cyclin-
dependent kinase activity through the signaling cascades initial-
ized by interactions between integrins and extracellular matrix
(11–15). For industrial production in bioreactors, the required
nfluenza-related illnesses cause an estimated 100,000 hospi-
talizations and tens of thousands of deaths in the United States
surface area can be provided using microcarrier beads (16–19).
Although this approach is sufficient to obtain high virus pro-
duction yield (18, 19), this propagation strategy is cumbersome
compared with propagation of cells in suspension. An MDCK
the scale-up process of influenza virus production.
In a previous study we compared the transcription profiles of
anchorage-dependent and anchorage-independent HeLa cells
using DNA microarrays (20). The gene siat7e (ST6GalNac V)
was identified as one of the genes that play a role in controlling
the degree of cell adhesion. It was shown that higher siat7e
transcription corresponded to a lower degree of adhesion by
microscopic evaluation and by monitoring cell detachment in a
siRNA was followed by enhanced adhesion. The human sialyl-
transferase ST6GalNac V, a member of the ST6GalNac family
of sialyltransferases, is a type II Golgi membrane protein that
transfers sialic acid from the donor CMP-Neu5Ac to the GalNac
residue on the ganglioside, GM1b, forming GD1?. Tsuchida et
al. (21) proposed indirect involvement of siat7e in synthesizing
disialyl Lea, a carbohydrate structure conjugated to proteins and
ceramides on the cell surface. In other studies, glycosphingolip-
ids including gangliosides have been reported to mediate cell
adhesion through the sugar residue interactions in the glycosyn-
apse microdomains (22, 23). These reports are consistent with
our findings on the relationship between siat7e gene expression
producers of several viruses including influenza A and B viruses.
The conversion of these anchorage-dependent cells to cells
capable of growing in suspension will simplify the production
process and has the potential to supplant current production
procedures in chicken embryonated eggs. In the present work we
report on the transfection of the anchorage-dependent MDCK
cells with the human siat7e gene, on the properties of the
siat7e-expressing cells and on their capability to produce the
Transfection of MDCK Cells with Human siat7e and Its Effects on
Cell–Cell Adhesion and Cell Spreading. Anchorage-dependent
MDCK cells exhibited changes in cell-cell adhesion and cell
spreading behavior following the incorporation of the human
siat7e gene as shown in Fig. 1. Cells transfected with the siat7e
shown in Fig. 1B (clone 1) and C (clone 2) appear to spread less
on the cell culture flask than the parental cells shown in Fig. 1A;
the siat7e-expressing cells also lost their ability to form a tight
Author contributions: C.C., M.B., and J.S. designed research; C.C. and V.L. performed
research; C.C., V.L., H.G., M.B., and J.S. analyzed data; and C.C., V.L., and J.S. wrote the
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
September 1, 2009 ?
vol. 106 ?
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