Materials Sciences and Applications, 2010, 1, 261-266
doi:10.4236/msa.2010.15038 Published Online November 2010 (
Copyright © 2010 SciRes. MSA
The Growth Study of Vero Cells in Different Type
of Microcarrier
Yusilawati Ahmad Nor1, Nurul Hafizah Sulong1, Maizirwan Mel1, Hamzah Mohd Salleh2, Iis Sopyan3
1Bioprocess and Molecular Engineering Research Unit; 2Halal Industry Research Centre; 3Biomedical Engineering Research Group,
International Islamic University Malaysia, Kuala Lumpur.
Received June 10th, 2010; revised October 4th, 2010; accepted October 5th, 2010.
The fact of microcarrier (MC) culture introduces new possibilities and makes possible the practical high-yield culture
of anchorage-dependent cells has generated a considerable focus in this study. The objective of this research was to
study the comparison of Vero cell growth on different types of commercial microcarriers; Cytodex-1, Cytodex-3,
Hillex® II and Plastic Plus in spinner vessel and two liters bioreactor cultured for 96 hours. Biological performance of
the microcarrier in RPMI media showed the preference of Vero cell grew on Cytodex 3 microcarriers with highest
maximum viable cell number (2.4
× 105 cells/ml) followed by Cytodex 1, Hillex and Plustic Plus. Vero cell on Cyto-
dex-3 data in spinner flask was compared in bioreactor and result showed higher viable cell number in biorector. Thus,
this dextran-crosslink gelatin microcarrier (Cytodex 3) provided the best surface for cell attachment and fast prolifera-
tion. At the end of this cell growth improvement will be used for virus transfection producing a vaccine in bioreactor.
Keywords: Cytodex 3, Microcarrier, Spinner Flask, Vero Cells, Vaccines
1. Introduction
Vero cell line was derived from the kidney of a normal,
adult, African green monkey (Cercopithecus) [1]. Vero
cells have been extensively used for producing viral vac-
cines [2-4] and for evaluating the performance of animal
cell in bioreactors with modified condition [5]. This cell
line also has been used extensively for virus replication
studies and plaque assays [1]. Moreover, Vero cells do
not disturb human health when used as substrate for bio-
logical product since they are free from oncogenic prop-
erty [2,6]. However, Vero cells are anchorage dependent
cells which required solid substrate to attach and grow.
Thus, Vero cells can be used in microcarrier and suspen-
sion cultures for large scale production in bioreactors.
The interaction between the cells and the substrate sur-
face is critical where cell adhesion occurs by divalent
cation and basic protein which occur between the solid
surface and the cell membrane [7]. Under proper condi-
tions, cells attach and spread onto the carriers and gradu-
ally grow out to a confluent monolayer [8].
Microcarrier culture comprises the cultivation of an-
chorage dependent cells on small solid substrate sus-
pended in growth medium and thus can be cultured in
larger scale. By using microcarriers in simple suspension
culture systems, it is possible to achieve yields of several
million cells per milliliter [9]. The microcarrier bioreac-
tor culture system offers an attractive method for cell
amplification and enhancement of phenotype expression.
Besides serving as substrates for the propagation of an-
chorage-dependent cells, microcarriers can also be used
to deliver the expanded undifferentiated or differentiated
cells to the site of the defect [10]. Microcarrier culture
also provides a method for rapid scaling-up with mini-
mum number subculture steps [11]. However, cell differs
in their attachment requirements, and thus a range of
microcarriers should be assessed for suitability. There are
various kinds of microcarriers based on material that
were used.
2. Experimental Details
2.1. Cell lines
Vero cells (African green monkey kidney cells) were
obtained from ATTC (CCL-81™) Cells were kept cryo-
preserved in liquid nitrogen until further use.
2.2. Culture Media and Chemicals
RPMI ( 50-020-PB) and RPMI without phenol red
The Growth Study of Vero Cells in Different Type of Microcarrier
Copyright © 2010 SciRes. MSA
( 90-022-PB) were supplied by Cellgrow. Fetal
Bovine Serum (FBS) (Cat. no 10270-098) obtained from
Invitrogen and Accutase (Cat. no AT104) from Innova-
tive Cell Technologies. Four different commercial mi-
crocarrier (MCs) were used which are Hillex® (Model
no H112-170) and Plustic Plus (Model no PP102-1521)
from Solohill (Michigan, USA); Cytodex 1 and Cytodex
3 from Amersham Biosciences (Uppsala, Sweeden).
MCs were prepared for use as recommended by the ma-
nufacturers. All other chemicals were obtained from
Sigma (St. Louis, USA).
2.3. Preparation of Microcarrier (MCs)
Cytodex 1, Cytodex 3, Hillex® and Plustic Plus micro-
carriers were prepared and sterilized according to manu-
facturer’s instructions. For Cytodex 1 and Cytodex 3
with each 3 g/l, the microcarriers were swollen with Ca2+,
Mg2+-free PBS twice and autoclaved at 115oC for 15
minutes while Hillex® (14 g/l) and Plustic Plus (20 g/l)
microcarrier were washed with deionized water and
autoclaved at 121oC for 30 minutes. All microcarriers
where incubated with the RPMI media before used.
2.4. Cell Dissociation
Media was discarded and cells were washed twice with
phosphate buffer saline (PBS), 2 ml Accutase enzyme
were added to a 75 cm2 flask and cells were incubated for
2 minutes at 37oC. Dislodged cells were then pooled with
RPMI media contained 10% serum and transferred into
inoculums flasks.
2.5. Spinner Flask Culture
Cultures were carried out in 250 ml spinner flasks
(Bellco Biotechnology, U.S.A) containing 200 ml of cul-
tured cells, at 37oC in a 5% CO2 incubator. The stirring
speed was maintained at 30 rpm at initial pH of the me-
dia adjusted to 7. The spinners were inoculated with 2 ×
105 cells/ml. The experiments were carried for 96 hours
and sampling was done every 8 hours.
2.6. Bioreactor Culture
The cultures were performed in a two liters bioreactor
(Labfors 3, Switzerland) containing one liter total work-
ing volume, equipped with a marine impeller. For start-
ing the microcarrier cultures, RPMI media was mixed
with 3 g/l Cytodex 3 in the bioreactor and incubated for 3
hours with starting volume of 900ml. Vero cells were
detached from T-flasks using PBS and Accutase enzyme
and 10% percent inoculums was added to the bioreactor
culture. The culture was seeded with 2.5 × 105 cells/ml
and was continuously agitated at 70 rpm. During cell
culture proliferation step, the following conditions were
applied: pH set at 7.2; pO2 maintained at 30% air-satura-
tion by injecting air or pure oxygen when required, tem-
perature at 37oC and agitation rate at 70 rpm. The ex-
periments were carried for 96 hours and sampling was
done every 8 hours.
2.7. Viable Cell Number (VCN) Counting
Two milliliters of Vero cell culture were washed twice
with PBS containing 0.02% (w/v) EDTA at pH7.6. PBS
was replaced with 0.25% (w/v) trypsin in Ca2+, Mg2+-free
PBS and EDTA (0.02%, w/v) and the tube was incubated
at 37°C for 15 minutes with occasional agitation. Two
mililiter culture medium containing 5% serum was added
to the microcarriers and centrifuged at 300 g and 4°C for
5 minutes. The supernatant is discarded and the pellet is
re-suspended in 2 ml Ca2+, Mg2+-free PBS containing
0.05% (w/v) tryphan blue. The concentration of cells in
the suspension is counted using haemocytometer.
2.8. Calculation
The specific growth rate μ (h1) calculation was esti-
mated by the following equation:
Inln n
where μ (day1) corresponds to the value of specific
growth rate at any given time point, t (hours) the culture
time and X (cells) the value of viable cell number for a
specific t. The doubling time, td (hour) calculation was
estimated by the following equation:
In 2
2.9. Monitoring Microcarrier
Samples of Vero cells culture were examined micro-
scopically using an inverted light microscope (Olympus,
Japan). Five mililiters Vero culture was transferred into
petri dish and was examined at three different micro-
scopic fields per sample.
2.10. Substrate Analysis
Sample was centrifuged at 28,000 rpm and 4oC for 15
minutes to remove cells and other contaminant. Super-
natant was collected and filtered using 0.45 micron filter
to remove any remaining particles. Glucose concentra-
tions were monitored by High Performance Liquid
Chromatography (HPLC) using SUPELCOGEL C-610H
column equipped with a refractive index and 210 nm UV
detector. Separation was done at 30oC, eluted at 0.5
ml/min using 0.1% H3PO4 and each sample was analyzed
for 30 minutes.
The Growth Study of Vero Cells in Different Type of Microcarrier
Copyright © 2010 SciRes. MSA
3. Results
3.1. Growth Kinetic of Vero Cells in Spinner
Flask and Bioreactor Culture
There were four runs in this experiment. Four types of
different commercial microcarriers (MCs) were used as
the substrate in this study which was Cytodex1, Cyto-
dex3, Hillex® and Plastic Plus (PP). Figure 1 below
showed growth profile of Vero cells in spinner flask on
different MCs while Figure 2 determined percentage
viability of the cells in the culture. The highest cell den-
sity was obtained at hour 88 and was equal to 2.4 ×
105cells/ml for 3 g/l Cytodex 3. Cells showed an expo-
nential growth for the period of 88 hour for Cytodex 3
and viable cells number decreased at hour 96. Cytodex 1
with the amount of 3 g/l was still increased at the end of
96 hour period with absence of lag phase for both Cyto-
dex 1 and Cytodex 3. Cytodex 1 reached the highest vi-
able cell number of 1.5 × 105 cells/ml followed by Hillex®
which reached 1.35 × 105 cells/ml and PP reached maxi-
mum cell number of 8.0 × 104 cells/ml, respectively. Vero-
Figure 1. Comparison viable cell numbers of Vero cell har-
vested from four types of MCs in spinner flask for 96 hour
period using RPMI growth medium supplemented with
10% FBS.
Figure 2. Percentage of cell viability in spinner vessel cul-
adhered slower to Plastic plus (PP) and Hillex® MCs
with the existence of lag phase at initial 8 hours. Besides
that, the cells was easily detached from these polystyrene
core substrate (Hillex® and PP) after 80 hours in
Hillex® MCs culture and reached stationary phase before
dropped in cells number at 88 hour.
From Figure 2 above, percentage of viability of each
MC was calculated. It was observed that average per-
centage of viability is 70 ± 30 for all MCs. Data for per-
centage viability for each MC was summarized in Table
1. Cells viability was highest in Cytodex 3 culture and
lower in Hillex and PP culture. However, the range of
viability indicated the viability is more that 50%. Thus,
this indicates the suitability of the condition parameter in
spinner vessel for the cells to grow.
Calculation of specific growth rate and doubling time
for four different MCs in spinner flasks was obtained
using Equations (1) and (2). Average specific growth (µ)
rate was determined at acceleration phase and Hillex®
has the highest specific growth rate which is 0.087h-1 and
lowest doubling time; 7.97 hour followed by PP (µ =
0.046 h-1, td = 15.1 hour), Cytodex 3 (µ = 0.022 h-1; td =
31.5 hour) and Cytodex 1 (µ = 0.022 h-1; td = 31.5 hour).
The results for Vero cells growth kinetic on each MC are
summarized in Table 2. From the data, Cytodex 3 was
used for scaling up Vero cell to two liters bioreactor and
the performance of the bioreactor was compared to spin-
ner vessel culture. Although Hillex® MC obtained the
lowest doubling time which is 7.97 hour; it was not se-
lected since the attachment of cells is slower and suscep-
tible to cells detachment.
Cell started to grow immediately after bioreactor in-
oculation. The maximal cell density level in bioreactor
was equal to 7.5 × 105cells/ml and was reached at 72 hour
after the start of the culture and the average specific
growth rate increased and equal to 0.031 h1 compared to
Table 1. Summarized percentage (%) cell viability in spin-
ner vessel culture.
MCs Cytodex 1 Cytodex 3 Hillex® PP
Viability 85 ± 8 90 ± 6 77 ± 15 77 ± 11
Table 2. Growth study of four different MCs in spinner
Responses Cytodex 1Cytodex 3 Hillex® Plastic
Plus (PP)
Maximum Cell no
1.55 ×
2.40 ×
1.35 ×
8.0 ×
Avg. Specific
growth rate (µ),
0.022 0.022 0.087 0.046
doubling time (td),
hour 31.5 31.5 7.97 15.1
The Growth Study of Vero Cells in Different Type of Microcarrier
Copyright © 2010 SciRes. MSA
Cytodex 3 culture in spinner flask. Cells number was
rapidly increased in the period of 48 hour and 56 hour
where the number of viable cells was doubled. Figure 3
shows the absence of lag phase on both spinner vessel
and bioreactor culture. However, the cells detached ear-
lier in bioreactor culture at period of 72 hour while de-
tached later in spinner vessel culture at 80 hour with the
absence of the stationary phase. From Figure 4, doubling
time of Vero cell was obtained as 22.4 hour compared to
31.5 hour in spinner vessel.
3.2. Glucose Uptake in MCs Culture
Regarding substrates glucose and metabolites profiles,
glucose level consumed by the cells in MCs culture was
observed during the cell growth phase and the results
showed in Figure 5 and Figure 6. Glucose concentration
was measured to infer the growth of cells in the MCs
culture and the sample was analyzed for every 8 hours.
Glucose concentration pattern was in decreasing order.
Figure 5 showed that glucose consumption was rapid
in Cytodex 1 and Cytodex 3 in spinner vessel culture.
Figure 3. Growth profile of Vero cell culture in spinner
flask and bioreactor using Cytodex 3 for period of 96 hours.
Figure 4. Specific growth rate of Cytodex 3 microcarrier
obtained in bioreactor.
Figure 5. Glucose uptake by Vero cell in spinner flask cul-
Glucose uptake rate was higher for Cytodex 1 and Cyto-
dex 3 for initial 8 hours and reduced gradually with time.
However, glucose uptake was slower in Hillex and PP
MCs culture and this explained the existence of lag phase
period of these polystyrene MCs culture. Final glucose
level of RPMI media was reduced to 0.517 g/l in Cyto-
dex 3 culture, 0.291 g/l in Cytodex 1, 0.4 g/l in PP and
0.061 g/l in Hillex® culture with initial glucose level of 2
g/l. Figure 6 shows glucose uptake in bioreactor and
spinner flask culture. Glucose uptake was slightly in-
creased at initial culture which reached to 2.48 g/l and
rapidly decreases to final glucose concentration of 0.4 g/l.
However, glucose concentration in spinner flask was
reduced consistently with time from initial glucose con-
centration of 2 g/l to 0.093 g/l. The glucose result shows
that total consumption of glucose in bioreactor was re-
duced compared to spinner flask culture; but with better
cells attachment.
3.3. Cell Distributions and Morphologies on MC
Observations under fluorescence microscope confirmed
the existence of viable Vero cells on the surfaces of the
Cell distribution on the microspheres shows in Figure
7 was observed after cultured for 80 h in spinner vessel.
Cytodex 1 and Cytodex 3 MCs were fully covered by
Vero cells where the cells were grown in multilayer and
the cells distributed more densely on the Cytodex MCs,
especially on the gelatin crossliked dextran (Cytodex 3).
This is consistent with the cell proliferation and viability
results in Figure 1. However, cells on PP MCs more
difficult to be observed compared to other three MCs.
Black region of Hillex® and PP MCs restricted cells
visibility. In addition cells were bridge between these
polystyrene core microspheres with elongated shapes. As
a result, these MCs were bound together and were not
easy to be separated. Moreover, only few separate cells
were observed on PP MCs thus confirmed with the
The Growth Study of Vero Cells in Different Type of Microcarrier
Copyright © 2010 SciRes. MSA
Figure 6. Glucose uptake of Vero cell using Cytodex 3 in
bioreactor for period of 96 hours.
b a
c d
Figure 7. Cell Attachment of Vero cell on four different mi-
crocarriers in spinner flask after 80 hours viewed under
electron scanning microscope at magnification of 20X; a)
Cytodex 1; b) Cytodex 3; c) Hillex®; d) Plastic Plus (PP).
growth profile of Vero in PP MCs. It was also observed
that higher densities of cells on Cytodex 3 MCs in biore-
actor compared to spinner vessel since higher maximum
cell recovery was obtained.
4. Discussions
Results showed that the selection of the proper MCs is
important to successful culture yield. In this study, cell
growth was examined on four types of easily available
MCs. The Vero cells adapted better to gelatin crosslinked
dextran (Cytodex 3) in spinner flask suspension culture
compared to other types of MCs. Cytodex 3 MCs yielded
the highest viable cell number compare to other MCs
(Figure 1) while PP and Hillex obtained lowest cell
number with minimum cell recovery. This is because of
the recovery of trypsin-released cells from the polysty-
rene MCs (Hillex® and PP) was more difficult and com-
plicated [12], since lower density made sedimentation of
MCs failed to adequately separate the cell polystyrene
MC mixtures. Poor recovery of cells from the polysty-
rene MCs may partially account for the reduced cell
counts reported here.
Furthermore, Cytodex 1 and Cytodex 3 have good mi-
croscopic ability compared to other MCs under study
(Figure 7). Because of the opacity of the polystyrene
MCs, the extent of cell attachment and growth was more
difficult to determine microscopically. The advantage of
Cytodex 1 and Cytodex 3 contributed easy monitoring
cell growing behavior on MCs. Moreover, cells harvest-
ing was easy for Cytodex 1 and Cytodex 3 since they
readily degradable compared to polystyrene which is
non-degradable [13]. Glucose data in Figure 5 analyzed
consumption of glucose by the cells and it showed that
glucose uptake was higher in Cytodex 3 culture com-
pared to other types of MCs. It was also showed that
glucose consumption was higher with higher adhered cell
number since the cells consumed glucose and converted
to energy that was used to attach on the surface of mi-
crocarriers as well as to support their live and growth
[14]. Figure 6 however shows reduced glucose uptake in
properly controlled bioreactor compared to spinner flask
culture. Increase of glucose concentration at initial cul-
ture in bioreactor was expected due to serum containing
glucose and thus subject to non-homogenous mixture
during sampling.
Hillex® has highest specific growth rate which is
0.087 h-1 and lowest doubling time (7.97 h) compared to
other MCs. However, they have lower maximum cell
number than Cytodex MCs since adaption of the cells in
this environment was slow. However, initial events that
occur when cell adhere to surface is critical because ad-
hesion precedes cell seeding and migration [15]. This
fact explained for lower cells number obtained during the
experiment. Furthermore, it was reported by Solo Hill
Inc. [16] that Vero cells were not preferred to grow on
plastic substrate. Results showed increasing viable cell
number in bioreactor from Figure 3 compared to spinner
flask culture. The results proved the suitability of Cyto-
dex 3 for upscaling Vero cells from spinner flasks to
bioreactor since critical parameters such as temperature,
pH and pO2 was properly control in bioreactor. Thus,
results show the compatibility of Vero cells for Cytodex
MCs. Thus, results are consistent with other study where
Cytodex™ MCs was widely used for Vero cell culture in
bioreactor. Furthermore, Cytodex 3 has been reported to
have the advantages of can be reused [17,18].
5. Conclusions
There are advantages and disadvantage relating to each
MCs culture system which has to be evaluated depends
The Growth Study of Vero Cells in Different Type of Microcarrier
Copyright © 2010 SciRes. MSA
on the purpose of the culture. In this study, Cytodex 3 in
spinner flask give the best performance to be used with
Vero cells culture compared to other types of MCs under
study. MC achieved even higher cell concentrations in
suspension of controlled bioreactors. Thus, Cytodex 3
believed to provide efficient system for production of
larger scale high value products such as vaccine using
Vero cells.
6. Acknowledgements
Thanks to laboratory assistant from Analytic Laboratory
of Biotechnology Engineering of IIUM for his excellent
work. This work was financially supported by the Minis-
try of Science and Technology (MOSTI) Malaysia.
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