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; and bBiotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, and cCenter 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 inuenza 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 identied 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 asks 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 Inuenza 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 specic production of hemagglutinin (expressed in hemagglutination units per 106 cells) from the siat7e-expressing cells was approximately 20 times higher than the specic 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

nfluenza-related illnesses cause an estimated 100,000 hospitalizations and tens of thousands of deaths in the United States annually (1). In response to rapid antigenic drift in influenza viruses, the most effective approach taken has been the distribution of trivalent inactivated viral vaccines, which are traditionally 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 periods, and cumbersome operations have spurred exploration of alternatives. Among the potential alternatives for vaccine production, the use of characterized, immortalized cell lines (particularly MDCK, VERO, and PER.C6) has been investigated. These cell lines have been found to produce consistently high viral titers (38). 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 cyclindependent kinase activity through the signaling cascades initialized by interactions between integrins and extracellular matrix (1115). For industrial production in bioreactors, the required
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surface area can be provided using microcarrier beads (1619). Although this approach is sufficient to obtain high virus production yield (18, 19), this propagation strategy is cumbersome compared with propagation of cells in suspension. An MDCK cell line that can proliferate in suspension would greatly facilitate 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 shear flow chamber (20), and inhibiting siat7e transcription using siRNA was followed by enhanced adhesion. The human sialyltransferase 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, glycosphingolipids including gangliosides have been reported to mediate cell adhesion through the sugar residue interactions in the glycosynapse microdomains (22, 23). These reports are consistent with our findings on the relationship between siat7e gene expression and cell adhesion. As was indicated earlier, MDCK cells are good 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 influenza virus. Results
Transfection of MDCK Cells with Human siat7e and Its Effects on CellCell 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. 1 A; 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 paper. The authors declare no conict of interest. Freely available online through the PNAS open access option.
1To

whom correspondence should be addressed. E-mail: yossi@nih.gov.

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Fig. 2. mRNA expression of human siat7e and endogenous GAPDH in parental MDCK and in clones 1 and 2 of the siat7e-expressing cells. (A) End-point RT-PCR. (B) Real-time PCR.

cationized ferritin and negative charge sites on the cell surface would allow charge measurements on cell surfaces by quantifying the amount of bound ferritin molecules (25). The signal profiles from each cell line, with and without ferritin treatment, are shown in Fig. 3. Flow cytometric analysis showed a shift in the overall signal distribution of the siat7e-expressing cells (Fig. 3B). The shift indicates higher signal intensities emitted from the fluorescein (FITC), which should correspond to higher number of anionic sites on the membrane surface. No difference was observed when the ferritin was not present (Fig. 3A).
Growth Kinetics in Monolayer and Suspension of the Parental and siat7e-Expressing MDCK Cells. Growth, viability, glucose consump-

Fig. 1. Parental and siat7e-expressing MDCK cells grown in T asks. (A) Parental MDCK cells. (B) Clone 1, isolated from the siat7e-expressing pool. (C) Clone 2, isolated from the siat7e-expressing pool.

junctions with the neighboring cells. It was also observed that when the siat7e-expressing cells undergo prolonged culture, some cells would self-detach while maintaining their viability. Assessment of transfection efficiencies with the siat7e plasmid using the FACSCalibur machine showed that approximately 4% of MDCK cells were transfected 24 h after introducing the plasmid vector.
Gene Expression Differences Between the Parental and the siat7eExpressing MDCK Cells. The detection of the human siat7e mRNA

tion and lactate production of the parental and the siat7eexpressing MDCK cells grown as a monolayer in T flasks are shown in Fig. 4 AC and as suspension culture in Fig. 4 DF. The siat7e-expressing cells grew less than the parental cells in the T flask (Fig. 4A). Their density reached 7 104 cells/cm2 com5 cells/cm2 of the parental cells after 179 h of pared to 2 10 growth, although the percent viability of the cells was similar (Fig. 4B). Glucose consumption and lactate production in the two cell lines were similar until the siat7e-expressing cells approached peak density in the T flasks as shown in Fig. 4C.

Surface Charge Differences Between the Parental and the siat7eExpressing MDCK Cells. To assess cell surface differences between

the two cell lines, the cell surface charge was measured using FITC-labeled cationized ferritin (2426). Interactions between
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Fig. 3. FITC signal distribution obtained by FACS analysis of parental and siat7e-expressing MDCK cells with and without ferritin. (A) Without ferritin treatment; (B) with ferritin treatment. Parental MDCK cells (gray line), siat7eexpressing cells (black line).

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in the parental and the siat7e-expressing cells and the expression of a housekeeping gene (endogenous GAPDH) are seen in Fig. 2A. It is clear that there is expression of human siat7e in the transfected cells but no expression in the parental cells, while GADPH expression was detected in all samples. Real-time PCR was performed to quantify the expression of siat7e and the expression of the housekeeping gene in clones 1 and 2 (Fig. 2B). The increase in the siat7e expression was correlated with the degree of cell-cell adhesion and cell spreading of these two transfected clones seen in Fig. 1.

Fig. 4. Growth parameters of parental MDCK cells (open circles) and siat7e-expressing MDCK cells (open squares) in shake ask in suspension and in monolayer in T asks. T asks: (A) Growth in viable cell density (VCD). (B) Viability %. (C) Glucose consumption and lactate production (shaded) in g/L. Shake asks: (D) Growth in viable cell density (VCD). (E) Viability %. (F) Glucose consumption and lactate production (shaded) in g/L.

Opposite growth trends were observed when the two types of cells were propagated in shake flasks. The growth curve (Fig. 4D) demonstrates that siat7e-expressing cells were able to proliferate in suspension culture, whereas the parental cells could not. The siat7e-expressing cells grew exponentially to a concentration of 7 105 cells/mL. High viabilities (Fig. 4E) of the siat7e-expressing cells were seen throughout the 12-day growth. These cells were at least 90% viable while the viability of the parental MDCK cells declined steadily over the culture period. The glucose and lactate profiles shown in Fig. 4F indicate that parental MDCK cells consumed more glucose and produced more lactate than the siat7e-expressing cells especially at later culture times when cell densities were greater in the siat7eexpressing cells. Microscopic analysis at the end of the growth showed that the surviving parental MDCK cells were aggregated in large clumps, while the siat7e-expressing cells, on the other hand, appeared healthy and were suspended primarily as individual cells.
Influenza Virus Growth and HA Titer in Parental and siat7e-Expressing MDCK Cells. The yield of influenza virus in parental and siat7e-

Summarized in Table 1 are the highest values of both the viral and the HA titers. The values were obtained 36 to 48 h post infection in the case of the adherent cells and 2438 h in the case of cells grown in suspension. The viral infectivity titers, expressed as 50% egg infectious dose per mL (EID50/mL), were similar in three growth conditions: monolayer culture of the anchorage-dependent parental MDCK cells, monolayer culture of the siat7e-expressing cells and the siat7e-expressing cells grown in suspension. However, remarkable differences were observed for HA titers, expressed in hemagglutinating units (HAU). When calculated per 106 cells, 2,155 HAU was obtained from the parental MDCK cells, 8,606 HAU from the siat7eexpressing cells grown in monolayer, and 54,348 HAU from the siat7e-expressing cells grown in suspension in shake flasks. Shown in Fig. 5 is the cell viability of the infected siat7eexpressing cells grown in suspension and the HA titers over the time course of one representative kinetic experiment.
Virus Antigenic Stability During Replication in Parental MDCK Cells and siat7e-Expressing Cells. The effect of different cell substrates

expressing MDCK cells was evaluated by analysis of growth kinetics of a model virus B/Victoria/504/2000 per 106 cells.
Table 1. Virus titers in different cell substrates

on virus antigenic properties was evaluated in hemagglutination inhibition test (HAI). The HAI titers of three ferret sera that were infected with egg-grown reference virus B/Victoria/504/

Viral titer Substrate MDCK monolayer siat7e-expressing cells monolayer siat7e-expressing cells* suspension HAU/mL 1,810 5,120 40,960 EID50/mL, log10 8.35 6.90 7.87 0.17 0.12 0.12

Virus titer per 106 cells HAU 2,155 8,606 54,348 EID50, log10 8.42 7.12 8.00

Inuenza strain B/Victoria/504/2000 was used to infect the substrates between M.O.I.s of 1.0 and 2.0 TCID50. Hemagglutinin titers and infectious titers were measured using supernatant from whole cell lysate samples. *Cells were infected at 107/mL density in suspension culture and then diluted to 106/mL for propagation.

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Fig. 5. HA production (open squares) and cell viability following infection of siat7e-expressing MDCK cells with inuenza B virus (lled circles). Cell viability of siat7e-expressing MDCK cells without infection are also shown (open circles).

2000 were determined using the B/Victoria output virus from the parental MDCK cells and the siat7e-expressing MDCK cells grown either in monolayers or in suspension. The results are shown in Table 2. In all cases, the sera titers were within two-fold difference, demonstrating that cell-derived viruses were as antigenic as those obtained from the egg-derived reference virus. Direct DNA sequencing of RT-PCR products amplified from HA and NA (neuraminidase) viral gene segments, showed that the cell-derived viruses and the egg-derived reference virus had identical nucleotide sequences. These data demonstrate that replication of the virus in parental or siat7e-expressing cells did not alter the antigenic properties of the virus. Discussion In a previous study, we identified two genes that have a role in cell adhesion (20): siat7e, a type II membrane glycosylating sialyltransferase, and lama4 which encodes laminin 4, a member of the laminin family of glycoproteins. These two genes were identified following a comparison of gene transcription of two phenotypically distinct HeLa cells, anchorage-dependent and anchorageindependent. It was demonstrated that decreased expression of siat7e in the anchorage-independent HeLa cells, or enhanced expression of the lama4, resulted in greater aggregation and morphological changes compared with the untreated anchorageindependent HeLa cells. An opposite effect was observed when expression of the siat7e was increased and the lama4 expression was decreased in the anchorage-dependent HeLa cells. Engineering cell lines to improve biotechnology processes was one of our major aims of the previous study. One of the important commercial production processes that are still in need of a welldefined cell line is the production of influenza virus. Influenza virus
Table 2. HAI titers with viruses from different cell substrates
HAI titers Substrate Chicken eggs siat7e-expressing cells suspension siat7e-expressing cells monolayer MDCK monolayer Sera 1 128* 256 64 64 Sera 2 256 512 128 128 Sera 3 256 512 128 128

Sera were obtained from three ferrets 3 weeks after intranasal infection with egg-derived reference virus B/Victoria/504/2000. *Reciprocal of the highest dilution of serum capable of completely inhibiting HA activity of the respective virus. Data are from a single representative experiment. Cells were infected at 107/mL density in suspension culture and then diluted to 106/mL for propagation.

is currently being produced in embryonated eggs (27). Since the production in eggs is quite cumbersome and time consuming, replacing the embryonated eggs process with mammalian cells, especially MDCK cells, is an appealing option (4, 6, 8). Since MDCK cells are anchorage-dependent, the replacement of the embryonated eggs with these cells will introduce additional processing difficulty. Specifically, these processes often require inclusion of microcarriers, which are difficult to scale-up. Microcarriers require multiple additional processing steps including seeding of cells on a surface for subsequent growth (which may be limited by surface area), maintenance of the microcarriers in the bioreactor along with sufficient mixing to avoid mass transfer limitation to and on the beads, and separation of the product from the microcarriers. Not surprising, current production of monoclonal antibodies and most other biopharmaceuticals by mammalian culture utilizes cell lines such as Chinese Hamster Ovary (CHO), Baby Hamster Kidney (BHK), and NS0 adapted to suspension culture. Conversion of the MDCK cells to grow in suspension would considerably simplify the production process of influenza vaccine. Although the current state-of-art has already used suspension MDCK cell cultures to produce influenza vaccines (28), we report the targeted engineering of the MDCK cell lines to anchorage-independent culture while others relied on long serial passaging approach. Incorporation of the human siat7e gene into the MDCK cells, as demonstrated in this study resulted in their conversion to anchorage-independent cells. The cells grew well in suspension in shake flasks. The cultures reached a concentration of 7 105 cells/mL maintaining at least 90% viability throughout the growth period. The human gene was successfully incorporated and transcribed, (Fig. 2 A) modifying considerably the cell phenotype. By using CF-FITC it was possible to determine that there is a change in the net charge on the surface of the siat7e-expressing cells. The increased negative charge may be associated with the increased number of sialic acids moieties attached to the cell surface gangliosides by siat7e. Elevated negative charge of the cell surface may contribute to a decreased cell-to-surface adhesion and to electrostatic repulsion between cells, and thus allowing the cells to grow in suspension. Siat7e-expressing cells were not only able to grow in suspension and to produce identical virus as the one produced in embryonated eggs, their specific production of HA was about 20 times higher than the anchorage-dependent parental cells. Recent publications have reported on the expression of the human siat1 gene (ST6Gal I) in MDCK cells that was associated with an increase in 2,6-linked sialic acid on glycoproteins (2931). The researchers wanted to increase the number of 6-linked sialic acids on the cell surface to enhance influenza virus sensitivity to neuraminidase inhibitor, which is the key component of antiviral drugs for influenza. In addition, a two-log increase in the production of human influenza viruses in these cells was reported (29). Unlike ST6Gal I, ST6GalNac V expressed in this current study is responsible for adding sialic acid to the GalNac residue located on the side position of oligosaccharide chains instead of the common terminal position on the Gal residue. The infectivity with the B/Victoria/504/2000 of the siat7e-expressing cells measured as EID50/cell was found to be slightly lower than that of the parental cells but the HA production was found to be higher. In a seasonal influenza vaccine, a typical dose is composed of 15 g purified HA from each of the three selected influenza strains (H1N1, H3N2, and B). Since the vaccine is characterized by the amount of the HA, it is an additional benefit of these cells. In work conducted by Tree et al., production of influenza virus A strain in MDCK cells grown on various types of microcarriers were compared to chicken eggs. They reported 5.0 104 HAU/mL in cells grown on Cytodex I in spinner culture and 2.0 105 HAU/mL in chicken eggs. Based on this information, they estimated that 1,000 L MDCK cells grown on solid microcarriers would be equivalent to roughly 30,000 eggs, or 1 L
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would be equivalent to about 30 eggs. In another study, titer of 2.5 103 HAU/mL was obtained in a stirred tank reactor using Cytodex I microcarriers (16) and a maximum titer of approximately 4.0 104 HAU/mL was obtained in WAVE cellbags using Cytodex I microcarriers (18). It is important to note that in these studies the Influenza A virus strain was used. For the Influenza B strains, which was used in our study, the HA titers are commonly on the scale of 100 (32, 33). Based on the HA production capability, we estimate that 1 mL of culture containing 106 cells can produce approximately 40,000 HAU. Taking into consideration that the average production of HAU per egg is also around 40,000, 10-L culture of the siat7e-expressing MDCK cells can produce the equivalent amount of HAU produced in 10,000 embryonated eggs. These data demonstrate that the established siat7e-expressing MDCK cell line has the potential to significantly increase the efficiency of manufacture of influenza vaccines, and thus, quite possibly contributing to lower vaccine cost and wider availability to a greater number of recipients worldwide. In the immediate future, it is imperative that we take on a systems approach, as demonstrated elsewhere (34), to integrate strain improvement, upstream optimization, and downstream processing to further improve our production strategy. A fully developed and well-characterized cell substrate system would be advantageous not only economically but also presents a stronger case for approval by federal regulation agencies. Materials and Methods
Cell Line and Virus. Madin Darby Canine Kidney (MDCK) cells were acquired from American Type Culture Collection (Cat. No. CCL-34). The MDCK cells were grown in 37 C, 5% CO2 humid incubator using Minimal Essential Medium containing Earls salts and L-glutamine (Invitrogen) and supplemented with Fetal Bovine Serum (Invitrogen) to a nal concentration of 10%. Only cells growing in less than 20 passages were used for this study. Inuenza virus strain B/Victoria/504/2000 was obtained from the inuenza virus depository of the Center of Biologics Evaluations and Research, Food and Drugs Administration (Bethesda, MD). Establishment of Stable MDCK Cell Line Expressing siat7e. Escherichia coli DH5 competent cells (Invitrogen) were transformed with full-length human siat7e gene expression vector (Cat. No. EX-V1581-M03, Genecopoeia). The plasmids were puried using the QIAprep Spin Miniprep kit (Qiagen) and were used to transfect MDCK cells using Lipofectamine 2000 reagent under manufacturers protocol (Invitrogen). The transfection procedure was as follows: day 1: MDCK cells were seeded at 2 105 cells/well in a 24-well plate; day 2: 0.8 g plasmid DNA was mixed with 2.0 L Lipofectamine 2000 and incubated together with the cells in OptiMEM I medium (Invitrogen) for 4 h; the cells were than washed and suspended in growth medium; day 3: G418 was added to the growth medium at a nal concentration of 0.400 mg/mL, and the medium containing G418 (selective medium) was routinely replaced every 3 to 4 days for a period of 3 weeks. Stably transfected pool of siat7e-expressing cells were grown and banked. Finally, clones were isolated by limiting dilution in a 96-well plate. Gene Expression. RNA samples were isolated from parental MDCK cells and from clones of the siat7e-expressing cells using RNeasy Total RNA Isolation kit (Qiagen). SuperScript One-Step RT-PCR kit (Invitrogen) was used for the reverse transcription and for PCR amplication experiments in accordance to the manufacturers protocol, using the sense primer sequence 5 -ttactcgccacaagatgctg-3 and antisense primer sequence 5 -gcaccatgccataaacattg-3 . GAPDH was selected as the endogenous control gene and was amplied using sense primer sequence 5 -aacatcatccctgcttccac-3 and antisense primer sequence 5 -gaccacctggtcctcagtgt-3 . Briey: cDNA synthesis was performed at 50 C for 30 min, samples were incubated at 94 C for 2 min to hot-start the DNA Taq polymerase. The PCR amplication cycle consisted of denaturation at 94 C for 15 s annealing at 55 C for 30 s, and extending at 72 C for 10 s (14 s for the endogenous control). The target genes were amplied for 35 cycles with a nal extension at 72 C for 10 min. The end products were resolved on a 1% agarose gel at 130 V for 30 min and captured on the gel imager (Bio-Rad). Real-time PCR was performed using Power SYBR Green RNA-to-CT 1-Step Kit (Applied Biosystems) with the same primer sequences described above. Briey: cDNA samples were synthesized from 0.5 ng RNA sample and amplied under standard thermal cycler protocol (50 C for 2 min, 95 C for 10 min,

and 40 cycles of 95 C for 15 s and 60 C for 1 min). Target Ct values were averaged from replicates and fold changes were calculated against the endogenous control, GAPDH. Cationized Ferritin Binding Assay. Cationized ferrtin (Electron Microscopy Sciences) was conjugated with FITC using the FITC Protein Labeling kit (Pierce Biotechnology). Briey: cationized ferritin was dialyzed with the supplied borate buffer and incubated with FITC solution at room temperature for 1 h. Excess FITC dye was removed using a dialysis cassette (Pierce Biotechnology). 79.9 and Conjugated ferritin complex was quantied using E270 nm1% MW 750,000 for native ferritin and a correction factor of 0.3 for FITC whose max 494 nm. The calculated F/P ratio was approximately 12. Approximately 1 107 cells were detached from culture asks using Hanks-based cell dissociation buffer (Invitrogen) and washed with PBS before re-suspending in 1 mL PBS containing FITC-conjugated ferritin at 50 g/mL nal concentration (24 26). The mixture was incubated on a thermomixer at 4 C for 1 h and washed once with PBS. Cells were spun down and suspended in 1 mL cold PBS. The cells were immediately analyzed using the FACSCalibur ow cytometer. Growth Kinetics. For growth kinetics in anchorage-dependent manner, parental and siat7e-expressing MDCK cells were seeded at a concentration of 2 105 cells per one 25-cm2 culture ask; 21 asks were seeded for each cell line. Glucose and lactate concentrations were measured using the YSI 2700 Select biochemistry analyzer (YSI Life Sciences) and cell count was measured using Cedex (Innovatis AG). Measurements were taken daily from three asks. For growth kinetics in suspension culture, cells from each line were seeded at approximately 2 105 cell/mL in three 125-mL vented shake asks containing 30 mL serum-supplemented Dulbeccos Modied Eagles Medium (Invitrogen) and shaken at 90 RPM. Measurements were taken at 48 h intervals. Virus Growth Evaluations in Monolayer and Suspension Culture. Monolayer culture: Parental MDCK cells or siat7e-expressing cells were grown to conuency in 25-cm2 asks (Corning). After removal of the growth media, the cells were washed once with serum-free medium and the virus was added to each ask at a multiplicity of infection (MOI) of 2.0 TCID50 (50% tissue culture infectious dose). After adsorption for 1 h at 37 C, the cells were washed with serum-free medium, and 10 mL of growth medium (containing 10% FBS) were added. The infected cells were incubated at 33 C for the remainder of the experiment. Cell condition (appearance of cytopathogenic effect) was constantly monitored and samples were collected every 8 h for virus infectivity and hemagglutination (HA) titers determination. Suspension culture: siat7e-expressing cells grown in shake asks were concentrated by centrifugation (600 rcf for 5 min) and re-suspended in a serum-free medium at a density of 107 cells/mL. After infection with the inuenza virus at an MOI of 2.0 TCID50, the cell suspension was incubated at constant shaking at 37 C for 1 h. At this time, the cells were precipitated and suspended in DMEM supplemented with 10% FBS to a density of 106 cells/mL. The infected cells were incubated at 33 C in the same conditions for the remainder of the experiment; the controlled culture was treated in the same way but without addition of the virus. Samples were taken every 8 h during a period of 4 days and stored in aliquots at 70 C for virus infectivity titer and HA titer determination. Cell concentration, viability and metabolic parameters were monitored at each time point. Determination of Virus Yield. Virus growth and concentration were determined by infectivity titer in chicken embryonated eggs (EID50) and by HA titer using standard techniques described earlier (3537). Determination of Virus Stability During Replication in MDCK Cells. Antigenic properties of the progeny virus harvested from the parental or the siat7eexpressing cells (56 h post infection) were characterized by hemagglutination inhibition test (HAI test) using a set of three homologous ferret antisera specic to strain B/Victoria/504/2000. The HAI test was performed in 96-well plates (two replicates for each serum sample) using 0.5% chicken red blood cells in PBS (pH 7.2) (37). Two viruses were considered antigenically indistinguishable if the corresponding HAI titers did not exceed two-fold difference. In addition the nucleotide sequences of viral gene segments encoding viral surface glycoproteins, HA and NA, were determined by direct DNA-sequencing of the RT-PCR products and compared with those of the parental virus stock. ACKNOWLEDGMENTS. We thank Dr. Bruce Raaka for his assistance with the FACS measurements and Dr. Pratik Jaluria for his communications on technical aspects of the experiments and formulation of the manuscript. This work was supported by the Intramural program at the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health.

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PNAS

September 1, 2009

vol. 106

no. 35

14807

APPLIED BIOLOGICAL SCIENCES