Download Application of a Serum‐Free Medium for the Growth of Vero Cells

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Biotechnol. Prog. 2000, 16, 854−858
Application of a Serum-Free Medium for the Growth of Vero Cells
and the Production of Reovirus
M. Butler,*,† A. Burgener,† M. Patrick,‡ M. Berry,†,§ D. Moffatt,† N. Huzel,†
N. Barnabé,†,| and K. Coombs‡
Departments of Microbiology and Medical Microbiology, University of Manitoba, Winnipeg,
Manitoba R3T 2N2, Canada
Two strains of reovirus (serotype 1 Lang/TIL and serotype 3 Dearing/T3D) were
propagated in Vero cells grown in stationary or agitated cultures in a serum-free
medium, M-VSFM. Solid microcarriers (Cytodex-1) were used to support cell growth
in agitated cultures with a normal doubling time of 25 h. Cell yields of 1 × 106 cells/
mL were obtained from an inoculum of 2 × 105 cells/mL in 4 days in microcarrier
cultures. The growth profile and cell yield was not significantly different from serumsupplemented cultures. The virus titer increased by 3-4 orders of magnitude over a
culture period of 150 h. The maximum virus titer in stationary cultures reached >1
× 109 pfu/mL for both strains of reovirus in M-VSFM. M-VSFM also supported high
viral yields in microcarrier cultures. Both the specific productivity and final viral yield
was higher in M-VSFM than serum-supplemented cultures. The high viral productivity
suggests that this is a suitable system for the production of reovirus as an oncolytic
agent for human therapeutic use.
Introduction
The mammalian reoviruses are members of the orthoreovirus genus of the family Reoviridae. The Reoviridae
include a number of important human and animal
pathogens such as rotavirus (Kapikian and Chanock,
1996) and orbivirus (Roy, 1996). Rotavirus infection is
estimated to be directly responsible for 5-25 million
deaths each year, and despite intensive efforts (Fifth
Rotavirus Vaccine Workshop, 1996), an effective vaccine
for these agents remains elusive. Reovirus, the prototype
of this family, remains an effective model for studying
the characteristics of the double-stranded RNA viruses
(Tyler and Fields, 1996).
Recent work from the University of Calgary, which
gained a significant amount of international publicity,
was the discovery that reovirus could be used as a cancer
cure through its property as an oncolytic agent (Coffey
et al., 1998). In the experiments, they showed sufficient
tumor regression in animal models to suggest the possibilities of the development of a new type of anticancer
agent which has been termed oncolytic. This finding
suggests the need to produce large quantities of reovirus
for human therapeutic use.
The virus is routinely grown in either monolayer or
stirred cultures of mouse L929 fibroblasts (Nibert et al.,
1996). However, it has also been reported that reovirus
* Department of Microbiology, 118 Buller Bldg., University of
Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. Telephone:
204-474-6543. Fax: 204-474-7603. E-mail: [email protected].
† Department of Microbiology.
‡ Department of Medical Microbiology.
§ Present address: Department of Pharmacy, University of
Manitoba, Winnipeg, Manitoba R3T 2N2.
| Present address: Aventis Pasteur, 1755 Steeles Ave. West,
Willowdale, Ontario MAR 3T4.
10.1021/bp000110+ CCC: $19.00
will grow in a number of other cell lines, including Vero
and Chinese hamster ovary (Taber et al., 1976; Davis et
al., 1990).
Vero cells have been accepted for viral vaccine production under specified regulatory guidelines (WHO, 1987a,b)
and are presently used in the commercial manufacture
of human and animal rabies vaccine as well as human
polio vaccine (El-Karamany, 1987; Shevitz et al., 1990;
Beale, 1992). Vero cells have been shown to be good
substrates for the propagation of bovine vesicular stomatitis virus (Nikolai and Hu, 1992), herpes simplex
virus (HSV) (Griffiths et al., 1981; Griffiths and Thornton, 1982; Marique et al., 1993), and rabies virus (Mendonca et al., 1993). These processes involve the culture
of Vero cells on solid microcarriers (Cytodex-1) in large
bioreactors up to 1000 liters (Montagnon et al., 1981,
1984).
The production of Vero cells in a culture system for
reovirus propagation has been studied previously in our
laboratory (Berry et al., 1999). Microcarrier cultures
(Cytodex-1 or Cultispher-G) in a serum-based medium
were shown to support successfully the propagation of
two serotypes of reovirus (type-1 Lang (T1L) and type-3
Dearing (T3D)).
However, serum-containing culture systems are becoming undesirable for the large-scale production of
vaccines. There are a number of disadvantages of serum
supplementation including batch-to-batch variation in
composition, the high protein content which hinders
product purification, and the potential for viral, mycoplasma, or prion contamination. Furthermore, the recent
threat to human health caused by the undefined agents
of bovine spongiform encephalopathy (BSE) is likely to
limit the continued use of bovine serum in culture
processes used for the synthesis of health-care products
such as viral vaccines.
© 2000 American Chemical Society and American Institute of Chemical Engineers
Published on Web 09/21/2000
Biotechnol. Prog., 2000, Vol. 16, No. 5
We have recently developed a novel serum-free formulation (designated M-VSFM) for the growth of Vero and
other anchorage-dependent mammalian cell lines. The
media does not contain any animal products and has a
very low protein content, <90 µg/L. In this report, we
investigate the characteristics of this serum-free medium
in supporting Vero cell growth and in the propagation of
reovirus.
Materials and Methods
(1) Maintenance of Vero Cultures. Adaptation to
M-VSFM. The cells were passaged directly into M-VSFM
(modified Vero serum-free medium) from serum-containing media without any adaptation protocol. The maximal
growth rate and cell yield were obtained after a few
passages of this transfer.
Cultures. Cells were maintained in T-flasks (75 cm2)
at 37 °C with a 10% CO2 overlay. The cells were passaged
every 5 days with a starting inoculum of 2.5 × 104 cells/
cm2 with 25 mL of pre-warmed medium. Microcarrier
cultures were established in 100 mL spinner flasks
(Bellco) with 2 g/L Cytodex-1 (Amersham-Pharmacia) and
an inoculation of 2 × 105 cells/mL.
Cell Passaging. The cells were first washed with 10
mL of PBS-EDTA (0.25%) and then treated with 1.0 mL
of trypsin (0.25%). After approximately 10 min, the cells
dislodged from surface attachment, at which point 1.5
mL of trypsin inhibitor (0.25% w/v) was added with 7.5
mL PBS. The cells are then centrifuged and resuspended
in fresh media.
(2) Growth Determination. Microcarrier Cultures. Samples (1 mL) were taken from an agitated
culture and placed into 1.8 mL Eppendorf tubes. Microcarriers were allowed to settle, supernatants were discarded, and microcarriers with cells were washed with
PBS-EDTA and then resuspended in 0.95 mL of 0.25%
trypsin-EDTA to dislodge the cells. Each sample was
mixed with an equal volume of Trypan Blue and counted
with a hemocytometer.
(3) Media Preparation. M-VSFM was prepared as a
10× liquid concentrate. Samples of this medium are
available upon request (Department of Microbiology,
University of Manitoba). Complete growth medium was
prepared by the addition of 10 vol % of M-VSFM
concentrate to DMEM. Serum-based growth medium was
prepared by the addition of 10 vol % of fetal bovine serum
(FBS) to DMEM.
(4) Virus Infection. Multiwell Plate Cultures,
Replicate plates were infected by removing 90% of the
medium and adding the virus sample at a multiplicities
of infection (MOI) of 0.5 and 5.0. The plates were gently
rocked every 10 min for 1 h after infection. Fresh media
was added to the flasks and incubated at 37 °C for 10
days.
Microcarrier Cultures. The cultures were infected
by removing 90% of the medium and adding the virus
sample at a multiplicity of infection (MOI) of 5.0. The
flasks were agitated intermittently at 40 rpm for 1 min
every 10 min for 1 h. Fresh media was then added to the
flasks and then placed at 37 °C for 10 days. Flasks were
then set at a stir rate of 40 rpm.
(5) Virus Sampling. Multiwell Plate Cultures. Sets
of infected plates were removed from the 37 °C incubator
each day and frozen at -70 °C for viral plaque assays.
The samples were freeze-thawed three times to ensure
complete lysis of the cells to release the virus.
Microcarrier Cultures. Samples (1 mL) of an evenly
distributed culture were taken daily and stored at -70
855
°C until required. The samples were then freeze-thawed
three times to ensure complete lysis of the cells.
(6) Viral Plaque Assay. The viral titers were determined by plaque formation on monolayers of mouse L929
cells. The thrice freeze-thawed samples were serially
diluted in gel-saline and added to subconfluent layers
of the cells in 6-well cluster plates. The plates were left
for 1 h with occasional rocking to ensure viral infection.
The cells were then overlaid with 2 mL per well of a 1:1
v/v mixture of 2% Bacto-agar and complete 2× media
199 (Gibco) and incubated at 37 °C. The wells were
overlaid a second time with 2 mL per well of the complete
agar/199 3 days later. A final layer of medium 199 that
contained 1.0% agar and 0.04% neutral red was placed
into the wells on day 6 postinfection. Plaques were
counted 18-24 h after the last overlay.
Results
The Development of a Serum-Free Medium. Serum-free media formulations have not generally been
available for anchorage-dependent cells largely because
of the complex nutrient and micronutrient requirements
of these cells. Specific proteins are required to ensure the
effective attachment of the cells on to a surface prior to
cell spreading and growth. Further difficulties may be
encountered in serial passages in the low protein environment where the activity of trypsin may persist. The
medium (M-VSFM) described in this report was developed by incremental improvements over several years.
Two important criteria in developing the medium were
the establishment of a minimal protein content and the
use of nonanimal-derived components. The initial prototype medium contained a high protein content, but this
was gradually reduced as the cells were adapted to a
medium containing a minimal content of nonanimalderived proteins. The final medium formulation contains
90 µg/L of recombinant growth factors derived from
transformed bacteria as well as a range of other nonproteinacious cofactors, all of which are nonanimal-derived.
The media also contains a range of trace elements which
are essential for the long-term growth and stability of
the cells. An important protocol in allowing this transition of cells from a serum-based medium was the
trypsinization method described earlier for routine cell
passaging. This method minimizes the required quantity
of trypsin to a level that can easily be neutralized by
trypsin inhibitor in the absence of serum.
The medium developed was shown to be suitable for
the growth of a number of anchorage-dependent mammalian cell lines, including MDCK and BHK, as well as
Vero. Growth rates and cell yields for these cell lines were
comparable between the serum-free and serum-supplemented media.
Growth Profile of Vero Cells in M-VSFM. The
ability of M-VSFM to support Vero cell growth was shown
in microcarrier (Cytodex-1) cultures (Figure 1). The cells
were inoculated at a concentration of 2.0 × 105 cells/mL
with 2.0 g/L Cytodex-1 microcarriers in 100 mL spinner
flasks for both M-VSFM and 10% FBS (fetal bovine
serum)-supplemented DMEM media. The cells reached
a maximum density of 1.0 × 106 cells/mL within 4 days
after inoculation in M-VSFM. Cell-surface attachment
was maintained throughout the cultures, and there was
no evidence of cell aggregation.
Reovirus Production in Stationary Cultures. Reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D)
were propagated in 6-well cluster dishes on monolayers
of Vero cells grown in M-VSFM and 10% FBS-DMEM
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Biotechnol. Prog., 2000, Vol. 16, No. 5
Figure 1. Growth of Vero cells in M-VSFM (2) and 10% FBSsupplemented DMEM (b) in microcarrier culture. The cells were
grown in 100 mL spinner flasks incubated at 37 °C with a 10%
CO2 overlay. Cytodex-1 microcarriers were added at a concentration of 2.0 g/L. The cells were added at a concentration of
2.0 × 105 cells/mL.
media (Figure 2a,b). The Vero cells were grown to a
density of 2.0 × 105 cells/cm2 prior to infection with the
virus. The propagation of virus was monitored in cultures
infected with virus at different MOI’s (multiplicities of
infection of 0.5 and 5.0).
The production of the T1L strain is shown in Figure
2a. In both media types, an MOI of 5.0 gave a high yield
of virus. Yields in M-VSFM reached a maximal titer of 1
× 109 pfu/mL after 5 days, and cells in 10% FBS-DMEM
reached a maximal titer of 2.2 × 108 pfu/mL after 4 days.
At an MOI of 0.5, M-VSFM supported a maximum yield
of 1.6 × 108 pfu/mL (8 days), whereas 10% FBS-DMEM
supported a maximum yield of 1.1 × 107 pfu/mL (3 days).
The propagation of the T3D strain is shown in Figure
2b. Similar to the T1L strain, an MOI of 5.0 gave a high
yield of virus. M-VSFM gave a maximum titer of 1.9 ×
109 pfu/mL after 3 days, whereas 10% FBS-DMEM gave
a maximum titer of 1.2 × 109 pfu/mL. At an MOI of 0.5,
M-VSFM supported a maximum yield of 1.5 × 109 pfu/
mL after 4 days. At the same MOI, cells in 10% FBSDMEM reached 0.9 × 109 pfu/mL in 2 days.
Reovirus Production in Agitated Microcarrier
Cultures. The propagation of reovirus (T1L and T3D)
is shown in Figure 3a,b. Cells were grown in spinner
flasks containing 2.0 g/L Cytodex-1 microcarriers. The
flasks were inoculated with 2 × 105 cells/mL in both the
M-VSFM and 10% FBS-DMEM cultures. The cells were
infected with virus (Type 1 Lang and Type 3 Dearing)
when the cells reached a density of 1.0 × 106 cells/mL (4
days). The cells were infected at an MOI of 5.0 with a
stirring regime as outlined in the Materials and Methods
section. The viral yield was monitored over a period of 9
days after infection.
Production of the T1L strain is shown in Figure 3a. A
maximum titer was reached within 2 days for cells grown
in M-VSFM. The highest titer reached was 6.6 × 107 pfu/
mL, and then steadily decreased with time. The cells
grown in 10% FBS-DMEM gave a maximum titer of 3.8
× 107 pfu/mL, a little more than one-half that of MVSFM.
Figure 2. Reovirus production from Vero cells in M-VSFM and
10% FBS-DMEM in stationary culture. Reovirus strains (a)
Type 1 Lang (T1L) virus or (b) Type 3 Dearing (T3D) virus were
propagated in 6-well cluster dishes from monolayers of Vero cells
grown in M-VSFM (1, 3) or 10% FBS-DMEM media (b, O). The
cells were infected with an MOI of 0.5 (3, O) or 5.0 (2, b).
A similar production pattern was observed for the T3D
strain (Figure 3b). Here, M-VSFM supported significantly
higher titers of the virus, which reached a maximum
yield of 1.1 × 109 pfu/mL after 2 days. Cells grown in
10% FBS-DMEM reached a titer of 6.1 × 108 pfu/mL in
the same time period.
The final yield of the T3D strain (1.1 × 109 pfu/mL)
was higher than the T1L strain (6.6 × 107 pfu/mL) from
the cells grown in M-VSFM. This may have been due to
the initial viability of the type 1 Lang sample used to
infect the microcarrier cultures. This did not affect the
growth profile, however, as the maximum titer for both
strains was reached during the time when the cells were
at their highest density of 1.0 × 106 cells/mL.
Infectivity of Reovirus in M-VSFM and FBSDMEM Cultures. The initial viral uptake by the cells
was determined in both stationary and microcarrier
cultures containing either M-VSFM or serum-supplemented medium (Table 1). After viral infection of the
cultures, samples of culture supernatant were withdrawn
after 1 h, and the extracellular virus titer was measured.
This revealed significant differences between the serumfree and serum-supplemented cultures.
For stationary cultures containing M-VSFM and infected with the T1L strain, >97% of the extracellular
Biotechnol. Prog., 2000, Vol. 16, No. 5
857
Table 1. Comparison of Virus Uptake in M-VSFM and FBS-DMEM during the Initial Infection Perioda
M-VSFM virus titer (pfu/mL)c
reovirus serotype
culture type
T1L
stationary
T3D
microcarrier
stationary
microcarrier
FBS-DMEM virus titer (pfu/mL)
MOIb
initial
after 1 h
initial
after 1 h
0.5
5.0
5.0
0.5
5.0
5.0
3.8 × 105
3.8 × 106
5.0 × 106
3.8 × 105
3.8 × 106
5.0 × 106
1.1 × 104
9.8 × 104
2.3 × 106
7.3 × 103
7.5 × 104
2.5 × 106
3.8 × 105
3.8 × 106
5.0 × 106
3.8 × 105
3.8 × 106
5.0 × 106
5.0 × 104
6.3 × 105
4.3 × 106
1.2 × 105
1.2 × 106
4.0 × 106
aSamples
of the culture supernatant were taken after 1 hour of infection to determine the extracellular virus titer.
of infection. c PFU ) plaque forming units.
b
MOI ) multiplicity
Table 2. Comparison of Specific Productivity of
Reovirus (T1L and T3D) from Vero Cells Grown in
Microcarrier Cultures with M-VSFM or FBS-DMEMa
media
T1L specific prod.
pfu/cell day
T3D specific prod.
pfu/cell day
M-VSFM
FBS-DMEM
28.5
16.1
584
364
a The values were taken from the first 2 days postinfection,
when viral productivity was at a maximum.
that high of uptake rates and only reached an 85%
decrease in the extracellular viral titer during this period.
For the T3D strain, the extracellular viral titer decreased
by 98% during this period in M-VSFM, whereas only 68%
was taken up by the cells in FBS-DMEM.
Similar results were seen in the microcarrier cultures.
For the T1L strain, the extracellular titer decreased by
>50% in M-VSFM during the postinfection period and
only 14% in FBS-DMEM. For the T3D strain, the
extracellular titer decreased by 50% during the first hour
in M-VSFM, but only by 20% in FBS-DMEM.
Comparison of Specific Virus Yields between
M-VSFM and FBS-DMEM. Table 2 shows the specific
productivity (pfu/cell per day) of either strain of reovirus,
T1L and T3D, in both types of media. The values were
determined at the peak of productivity during the first 2
days postinfection. The viral productivity was significantly higher in cultures containing M-VSFM. For the
T1L strain, M-VSFM resulted in a 173% increase in viral
productivity per cell compared to cells grown in FBSDMEM. Similarly, for the T3D strain, cells in M-VSFM
were 175% more productive in generating the virus.
Discussion
Figure 3. Reovirus production in microcarrier cultures of Vero
cells in M-VSFM and 10% FBS-DMEM. The cells were grown
in 100 mL spinner flasks incubated at 37 °C with a 10% CO2
overlay. Cytodex-1 microcarriers were added at a concentration
of 2.0 g/L. The flasks were inoculated with 2 × 105 cells/mL in
both the M-VSFM and 10% FBS-DMEM cultures. The cells were
infected (indicated by arrow) with either (a) Type 1 Lang virus
or (b) Type 3 Dearing virus when the cells reached confluence
(4 days). The points show cell growth for M-VSFM (4) or 10%
FBS-DMEM (O) and viral titer for M-VSFM (2) or 10% FBSDMEM (b).
virus decreased during the first hour in M-VSFM.
However, cultures containing FBS-DMEM did not show
The serum-free medium (M-VSFM) which we have
developed supports the growth of the anchorage-dependent cell line Vero in stationary and agitated microcarrier
cultures. M-VSFM is free of any animal-derived components and has a low content of nonanimal-derived protein
(90 µg/L). Vero cells adapted directly to M-VSFM from
serum-based media. The growth profile of cells in MVSFM can be maintained for >10 passages in T-flasks.
The growth profile and cell yield was not significantly
different from serum-supplemented cultures having a
normal doubling time of 25 h.
The formulation can be prepared as a complete liquid
medium (1×) or as a concentrated (10×) serum-free
supplement to be added to a standard growth medium.
The growth characteristics of these cultures are superior
to alternative commercial media tested or reported in the
literature.
We have shown that two types of reovirus can be
propagated from our serum-free medium M-VSFM in
both stationary and agitated culture. The final viral yield
was higher in both types of culture systems over the yield
obtained from cells grown in serum-supplemented medium.
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Biotechnol. Prog., 2000, Vol. 16, No. 5
M-VSFM showed a clear difference in the specific
productivity of the virus in agitated culture. For the two
strains, a substantial increase in viral productivity was
found over the serum-containing system. Cells in MVSFM produced the virus >170% over cells grown with
serum. This suggests that the cells in M-VSFM may be
in a metabolic state more favorable for viral infection
than in the serum-supplemented medium. Indeed, serum
contains many undefined components that could hinder
the infectivity of the virus or possibly enhance the
cytopathic effects mediated by the virus. This is consistent with our observations of a sharper decline in the cell
viability in the serum-containing cultures.
Infectivity of reovirus seems to be hindered by the
presence of serum. The uptake of virus into the cells is
higher in M-VSFM than in traditional serum media. The
high protein content in serum may inhibit the infection
of the virus, or by some other means. This further
supports the advantage of serum-free formulations for
these types of processes.
The stability of reovirus was higher in M-VSFM than
in FBS-DMEM. In the microcarrier cultures, for both the
T3D and T1L strain, a sharper decline in the viral titer
was observed in FBS-DMEM. This would suggest that
serum contains viral inhibitors, such as proteases, that
decreases the stability of the virus. This further supports
a conclusion that M-VSFM is better suited for a largescale process.
The overall yield of the two strains was significantly
different in the microcarrier cultures. The maximum
yield of the type 1 Lang was only 6.6 × 107 pfu/mL,
whereas the type 3 Dearing strain reached titers of 1.1
× 109 pfu/mL. It is not known why there is a discrepancy
in the overall production yields. High viral titers have
been achieved in previous microcarrier cultures of Vero
cells in our lab (Berry et al., 1999).
In conclusion, M-VSFM is suitable for the production
of reovirus from Vero cell culture. M-VSFM is found to
be superior to serum-containing media in both cell growth
and virus production. Infection of the Vero cells with two
strains of reovirus (type 1 Lang and type 3 Dearing)
resulted in a high viral titer in M-VSFM. M-VSFM
supports the growth of these cells comparable to serumcontaining media, and it is suitable for production of high
yields of reovirus. Productivity of the virus is superior
in M-VSFM than serum-supplemented cultures. This
system is suitable for the development of a bioprocess
for the production of reovirus for human therapeutic use.
Acknowledgment
This work was supported by a grant from the Natural
Science and Engineering Research Council of Canada.
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Accepted for publication August 21, 2000.
BP000110+