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Rotary culture promotes the proliferation of MCF-7 cells encapsulated in three-dimensional

collagen–alginate hydrogels via activation of the ERK1/2-MAPK pathway

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2012 Biomed. Mater. 7 015003

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IOP PUBLISHING BIOMEDICAL MATERIALS
Biomed. Mater. 7 (2012) 015003 (10pp) doi:10.1088/1748-6041/7/1/015003

Rotary culture promotes the proliferation

of MCF-7 cells encapsulated in
three-dimensional collagen–alginate
hydrogels via activation of the
ERK1/2-MAPK pathway
Hongxia Zheng 1,6 , Weiming Tian 2,3,4,6 , Hongji Yan 1 , Lei Yue 2,3 ,
Yao Zhang 2,3 , Fengtong Han 2,3 , Xiongbiao Chen 5 and Yu Li 1,2,3,7
1
Department of Biological Science and Technology, Harbin Institute of Technology, Harbin, 150080,
People’s Republic of China
2
The Academy of Fundamental and Interdisciplinary Science, Harbin Institute of Technology, Harbin,
150080, People’s Republic of China
3
Department of Biological Science and Engineering, Harbin Institute of Technology, Harbin, 150080,
People’s Republic of China
4
State Key Laboratory of Space Medicine Fundamentals and Application, Chinese Astronaut Research
and Training Center, People’s Republic of China
5
Department of Mechanical Engineering, University of Saskatchewan, Saskatoon, Canada
E-mail: liyugene@hit.edu.cn

Received 1 October 2011

Accepted for publication 16 December 2011
Published 20 January 2012
Online at stacks.iop.org/BMM/7/015003

Abstract
Rotary cell culture systems (RCCS) have been shown to be promising for promoting
three-dimensional (3D) cell growth and assembly of cells into functional tissues. In this study,
3D tissue-like spheroids of MCF-7 cells were constructed by encapsulating the cells in the
collagen–alginate hydrogel, and then cultured in a RCCS to investigate the proliferation of
MCF-7 cells. The results from the MTT assay showed that the proliferation rate of MCF-7
cells cultured in the RCCS was higher than that of the static culture control group, and the
results from the flow cytometry revealed that the cells in S and G2/M phase were significantly
increased compared to the control group. The expression of cell proliferation antigen PCNA
and cyclin D1 was also examined with the results further supporting the enhanced proliferation
of MCF-7 cells by the RCCS. The results from indirect immunofluorescence revealed that the
rotary culture altered neither the cytoskeleton distribution nor the assembly of mitotic spindle.
By examination, it was also shown that the rotary culture induced the ERK1/2-MAPK
pathway. Taken together, this study demonstrated that the rotary culture could promote the
proliferation of MCF-7 cells by inducing the ERK1/2 pathway.
(Some figures may appear in colour only in the online journal)

1. Introduction

6 These authors contributed equally to this paper. Breast cancer is among the common causes of death for
7 Author to whom any correspondence should be addressed. women around the world. In order to reduce the rate of death

1748-6041/12/015003+10$33.00 1 © 2012 IOP Publishing Ltd Printed in the UK & the USA
Biomed. Mater. 7 (2012) 015003
resulting from breast cancer, researchers have made progress
in discovering preventive therapies for this common cancer
[1]. Up to the present, most of the preclinical therapeutic
development for carcinomas in vitro has relied on traditional
two-dimensional (2D) cell culture systems. However, previous
studies [2, 3] show that 2D culture models may not be
able to examine the relative resistance of tumor cells to
chemotherapeutic drugs, which is often found in vivo, and
further suggest that three-dimensional (3D) culture models
may provide a better model for this examination. 3D culture
models can provide a cellular microenvironment that closely
mimics the biological features observed in native tissues,
including complex cell–cell and cell–matrix interactions
[3–6]. These features are critical to the cancer progression.
As such, 3D culture models are considered more suitable for
the development of new antitumor drugs and therapies for the
treatment of breast cancer.
By providing cells with a 3D environment, cell
encapsulation in hydrogel is one of the promising strategies
for tissue engineering and cellular therapy and has been
widely used to develop a 3D growth pattern of cells to
mimic tissue in vivo [7–10]. Various biomaterials including
collagen, fibrin, hyaluronic acid, alginate and gelatin have been
widely used for cell encapsulation [10–15] for their ability
to provide an extracellular matrix (ECM)-like environment.
Alginate hydrogel is among the most widely used due to its
biocompatibility and readily controlled gel process by dripping
a sodium alginate solution into an aqueous solution of calcium
ions typically formed from calcium chloride (CaCl2 ) [16].
Previous studies have demonstrated that alginate hydrogel
has been successfully used to deliver cells and proteins to
patients [16–19]. In our research, to create the in vivo-mimic
microenvironments, alginate hydrogels were made with the
addition of collagen I.
The 3D culture model of MCF-7 human breast cancer
cells was studied by Dhiman et al [20]. Compared to cells
cultured in 2D, MCF-7 cells in a 3D environment more
accurately represent the in vivo tumors [20]. However, the
limited diffusion of nutrients and metabolic products due to
the static 3D culture environments in [20] can prevent cells
growing as they might in vivo. The rotary cell culture system
(RCCS), pioneered by the National Aeronautics and Space
Administration in the 1990s, is a powerful tool to grow 3D
cell clusters in microgravity, in which cells are maintained in a
dynamic fluid suspension, thereby overcoming the drawbacks
associated with the static 3D culture systems [21–23]. The
simulated microgravity (SMG) environment of the RCCS can
be used to generate the macroscopic tissue environment, as
illustrated in a variety of basic and applied medical studies
[24–26]. As such, we selected the RCCS for use in the 3D
MCF-7 cell culture in this study.
In this study, 3D tissue-like spheroids of breast cancer
MCF-7 cells were constructed by encapsulating the cells in the
collagen–alginate (CA) hydrogel and then cultured in a RCCS.
With this model, the effect of rotary 3D culture on the MCF-7
cell proliferation and on the activation of the ERK1/ERK2-
MAPK pathway in cell culture was examined and evaluated.
2
H Zheng et al
2. Methods
2.1. Measurement of the mechanical properties of the
CA hydrogel
The CA hydrogel was formed with varying ratios of collagen
to alginate, specifically at 10:1, 20:1, 30:1, 40:1, and 50:1, in
which the alginate concentration was 0.01 mg ml−1 . It is known
that the mechanical properties of the cross-linked hydrogel are
of importance to the cell culture in the RCCS. In this study,
these properties were examined by means of a Bohlin Gemini
II rheometer (Malvern Instruments, UK) with the parallel plate
geometry (40 mm in diameter). Specifically, 5% deformation
amplitude was applied to the hydrogel samples, and the elastic
and viscous moduli were evaluated from the data recorded
by sweeping the frequency from 0.1 to 100 Hz. The cone
was covered with an adapted water trap to prevent water from
evaporating during the experiment.
2.2. 3D MCF-7 cell spheroid construction in the CA hydrogel
Human breast cancer cell line MCF-7, purchased from
American Type Culture Collection (Manassas, VA, USA),
was cultured in the DMEM culture medium with 10%
FBS (GIBCO) and 0.01 mg ml−1 bovine insulin in an
incubator with 37 ◦ C, 5% CO2 atmosphere and 99% humidity.
Collagen/alginate composite microspheres for the MCF-7 cell
culture were prepared as previously reported by Bucaro et al
[27]. Briefly, a 1% solution of low viscosity sodium alginate
(Sigma) in 30% collagen was prepared by stirring for several
hours. The solution obtained was sterilized by membrane
filtration (0.22 μm pore size) and then mixed with MCF-7
cells. Drops of the cell suspension in collagen/alginate were
gently delivered with a 23-gauge needle to a gelling bath
containing an isosmotic solution of calcium chloride. The
alginate droplets gelled once in contact with the calcium
chloride. With the adjustment of the electric field voltage,
beads were formed with diameters of approximately 1–3 mm,
and about 2 × 104 cells were encapsulated in each bead of
the CA hydrogel. The MCF-7 cell encapsulated composite
hydrogels were cultured in the medium for 48 h to construct
MCF-7 spheroids.
2.3. Microscope examination of the cell-like spheroids in the
CA hydrogel
The structure of the CA hydrogel with encapsulated
cells, once lyophilized, was examined using scanning
electron microscopy (SEM) (FEI QuANTA-200F, USA).
The morphology of the cells cultured in hydrogels was
examined with an inverted phase-contrast microscope (DP71,
OLYMPUS, Japan) and SEM using the method detailed in our
previous study [28].
2.4. Measurements of glucose concentration
The glucose concentration in the culture medium was
measured using a glucose (GO) assay kit (Sigma), and the
cell viability was examined using Vi-CELL XR (Beckman
Coulter).
Biomed. Mater. 7 (2012) 015003
2.5. Rotary culture
Rotary culture (or the rotating group) was performed with
a one-axis RCCS (Synthecon, USA) at 15 rpm. The high
aspect ratio vessel used in the system is filtered with a gas
permeable membrane to allow passive gas exchange. With
rotation, the RCCS can simulate a microgravity environment
with a gravity acceleration of 10−2 g [29], thus providing
preferable suspension culture conditions. The cells of control
group were cultured in a static environment. Both rotating and
control groups were cultured with 1% FBS.
2.6. Harvest of cells from the CA hydrogel
MCF-7 cells were isolated from the CA hydrogel by dissolving
the hydrogel in a solution of 25 mM sodium citrate, 10 mM
EDTA and 115 mM NaCl, with a pH of 7.4, for 15 min. MCF-7
spheroids were collected by centrifugation [30].
2.7. MTT assay for the proliferation of MCF-7
Cell proliferation was tested using a 3-(4,5-dimethylthiazol-
2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay (Sigma,
USA). At 24, 48, 72 and 96 h of cell culture, 10 μl of the
MTT solution was added to each well. After 4 h of incubation,
colored crystals of formazan were dissolved with 150 μl of
a dissolving solution (DMSO). Plates were kept on an orbital
shaker for 5 min and the absorbance was measured at 570 nm
with a microplate reader (Tecan Group Ltd, Infinite M200,
Switzerland). Each group has eight samples.
2.8. Cell cycle analysis
Flow cytometric analysis was performed to investigate the
effects of RCCS-rotary culture on MCF-7 cell cycle in this
study. After 48 h rotation, MCF-7 cell spheroids were collected
from the disassociation of the CA hydrogel mentioned in
section 2.6. The MCF-7 cells obtained from both rotating and
control groups were trypsinized, harvested and fixed in 1 ml
of 75% cold ethanol and then incubated at 4 ◦ C for 15 min,
respectively. After incubation, the cells were centrifuged at
800 rpm for 5 min and the cell pellets were then suspended
in 500 μl propodium iodine (50 μg ml−1 ) containing 100 μg
ml−1 RNase, 0.1% NP-40 and 0.1% sodium citrate, which
is followed by incubation on ice for 30 min. The cell cycle
distribution was evaluated from 20 000 cells using the ModFit
LTTM software (Becton Dickinson, USA) and the FACS
caliber (Becton Dickinson, USA).
2.9. Immunofluorescence staining
At 48 h of the MCF-7 cell culture, the cytoskeleton
(microfilament and microtubule) was evaluated by means
of the indirect immunofluorescence technique. Cells were
washed in phosphate-buffered saline (PBS) and subsequently
fixed with 4% paraformaldehyde in 0.1 M PBS for
15 min. Cells were then permeabilized for 5 min with
0.1% Triton X-100 in PBS and rinsed three times
with PBS. Next, the microfilament was stained with
3
H Zheng et al
25 μg ml−1 phalloidin-FITC (Sigma) to visualize F-actin.
Staining of microtubules was performed with 7.5 μg ml−1 anti-
α-tubulin antibodies (Sigma) incubated for 2 h at room
temperature. Cells were washed three times with PBS and
incubated for 1.5 h at room temperature with anti-mouse
TRITC-conjugated immunoglobulin antibody (Zhongshan
Golden Bride Biotechnology Corporation, Beijing, China).
Eventually, cells were counterstained with 1 μg ml−1 of DAPI
(Amresco) for 2 min and images were captured with a laser
confocal microscope (Carl Zeiss 510 LSM, Inc.).
2.10. Western blot
After rotating for 48 h, MCF-7 cell spheroids were also
collected and centrifuged at 800 rpm. The cell pellets were
collected and suspended in lysate buffer (50 mM tris-
HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium
deoxycholate, 0.1% SDS) containing protease inhibitors on
ice for 30 min. The lysate solution was centrifuged at
12 000 rpm for 10 min at 4 ◦ C and the supernatants were
collected. The protein concentration was detected using the
Bradford method. Specifically, proteins with the same amount
of 40 μg ml−1 were loaded on the lanes of 12% SDS-
PAGE gel via electrophoresis and then transferred to a
PVDF membrane via semidry transfer. The membrane was
blocked with 5% non-fat milk for 2 h at room temperature
and then incubated with antibodies against proliferating cell
nuclear antigen (PCNA) and cyclin D1 (Abcam) for the cell
proliferation antigen detection, anti-ERK1/2 (Santa), anti-
phospho-ERK1/2 (Santa), anti-JNK (Santa), anti-phospho-
JNK (Santa), anti-p38 (Santa), and anti-α-tubulin (Sigma) for
cell signaling protein expression. The specific protein band
was visualized with an ECL kit (Amersham biosciences).
2.11. Statistical analysis
All data are presented as the mean ± standard deviation,
determined from three or more runs of experiments. Statistical
analysis was performed by a two-tailed Student’s t-test and the
differences were considered insignificant if P < 0.05.
3. Results
3.1. Mechanical properties of the CA hydrogel
The mechanical properties of the CA hydrogel with varying
ratios of collagen/alginate were examined with a rheometer.
Figure 1 shows the elastic modulus and viscous modulus
evaluated from the measurements as a function of frequency.
It is seen that both moduli of the hydrogels with ratios of
30:1, 40:1 and 50:1 (collagen/alginate) exhibit a plateau in
the frequency range of 0.1–25 Hz, suggesting their mechanical
stability over this frequency range. It is also seen that for the
hydrogels with ratios of 30:1, 40:1 and 50:1, the elastic moduli
are larger than the viscous moduli. This indicates that the
hydrogel was well gelled from a viscous liquid. The viscous
modulus of the hydrogel increases with the frequency and
becomes larger than the elastic modulus at high frequencies,
depending on the ratio of collagen/alginate. In this study,
Biomed. Mater. 7 (2012) 015003
(a)
(c)
(e)
Figure 1. Measured mechanical properties of the CA hydrogel with varying ratios of collagen/alginate: (a) 50:1, (b) 40:1, (c) 30:1, (d) 20:1
and (e) 10:1. Both x- and y-axes are in log scale.
the CA hydrogel with a ratio of 30:1 collagen/alginate was
selected for encapsulation of cells.
3.2. MCF-7 cells encapsulated in the CA hydrogel
MCF-7 cells encapsulated in the CA hydrogel with a ratio of
30:1 collagen/alginate were examined by SEM with the results
shown in figure 2. Specifically, figure 2(a) shows the porous
structure of hydrogel, which allows for the rapid transportation
of nutrients and metabolic products; and figure 2(b) shows the
3D spheroid pattern. By the images from the phase-contrast
micrograph, figure 3(a) shows the distribution of MCF-7 cells
in the CA hydrogel right after encapsulation; and figure 3(b)
shows MCF-7 cells forming large and multi-cellular spheroids
at 48 h in static culture. The results from the cell viability
assay showed little change in cell viability between right after
encapsulation and at 48 h of cell culture, both around 93%.
Figure 4, as an example, shows camera images of cells right
after encapsulation and at 48 h of cell culture. In addition, we
4
H Zheng et al
(b)
(d)
measured the glucose concentration in the culture medium at
different time points using a Glucose Assay Kit. The results
are given in figure 5, showing that the glucose concentration
decreases gradually with time. Taken together, our results show
that the CA hydrogel with a ratio of 30:1 collagen/alginate is
able to provide a suitable environment for the growth of breast
cancer cells in vitro.
3.3. Rotary culture enhanced the proliferation of MCF-7 cells
encapsulated in the CA hydrogel
Although the CA hydrogel was shown to be able to provide
3D environment for MCF-7 cells, the diffusion of nutrients
and metabolic products might be limited by the static 3D
culture environments. In this study, the rotary culture system
was used for 3D cell culture, as compared to the static
control group. In order to remove interference of culture
serum, 1% FBS was used for cell culture in both groups.
The comparison results are shown in figure 6. Specifically,
Biomed. Mater. 7 (2012) 015003 H Zheng et al

(a) (b)

Figure 2. SEM images of the CA hydrogel with a ratio of 30:1: (a) porous structure of the hydrogel and (b) 3D MCF-7 cells in the hydrogel.
The arrows point to the cells in the hydrogel.

(a) (b)

Figure 3. Phase-contrast micrograph images of MCF-7 cells in the CA hydrogel at (a) 0 h and (b) 48 h. The arrows point to the cells seeded
in the hydrogel.

(a) (b)

Figure 4. Camera images of live/dead cells at (a) 0 h and (b) 48 h.

figure 6(a) shows the comparison of the MCF-7 cell growth
between the rotating group and the control group with the
MTT assay. The results show that MTT values in the rotating
group are significantly higher than that of the control group
at 48, 72 and 96 h, respectively. This suggests that rotary
culture can enhance the proliferation of MCF-7 cells in the CA
hydrogel.
Flow cytometric investigation was performed on the
samples that were cultured for 48 h in rotating and static
environments, respectively. The results are presented in
figure 6(b). It is seen that there is a significant difference in
cell cycles between the rotating and control groups, and that
the per cent of cells in S and G2/M phase after 48 h of rotation
increases significantly as compared to the control group, while
the per cent of cells in G0/G1 phase decreases (figure 6(b)).
In both groups, FACS analysis showed that the per cent of
apoptotic cells was not significant.
To confirm the ability of rotary culture to promote the 3D-
MCF-7 proliferation, we also studied the expression of PCNA
and cyclin D1 in both rotating and control groups with the
results shown in figure 6(c). The expression of both PCNA
and cyclin D1 was significantly elevated in the rotating group

5
Biomed. Mater. 7 (2012) 015003
Figure 5. Glucose concentration in the culture medium measured at
different time points. All data are presented as the mean ± standard
deviation of triplicate experiments (n = 3).
compared to the control group, which confirms the enhanced
proliferation in the rotating group.
Taken together, these results showed that rotary culture
could provide a more preferable condition for 3D-MCF-7 cells
than the static culture.
3.4. Rotary culture did not alter cytoskeleton of the
3D-MCF-7 cell distribution
Cytoskeleton is important to the regulation of proliferation,
as observed in the studies [31, 32] on the cell growth in the
2D microgravity environment. In this study, the distribution of
cytoskeleton was assessed with indirect immunofluorescence.
(a)
(b-1)
Figure 6. Comparison of the 3D-MCF-7 cell proliferation in the rotary culture environment and static condition: (a) the MCF-7 cell
proliferation evaluated by the MTT assay; (b) cell cycle analysis of MCF-7 cells by flow cytometry, specifically (b-1) shows the cell cycle in
histogram and (b-2) shows the per cent of cell number in cell cycle with the data expressed as the mean ± standard deviation of
experiments (n = 3, ∗ indicates that P < 0.05 and ∗∗ indicates that P < 0.01 as compared to the control group); and (c) PCNA and cyclin D1
expression with the western blot analysis.
6
H Zheng et al
The results are shown in figure 7, suggesting that the
cytoskeleton in both rotating and static groups has a similar
distribution. Specifically, figure 7(a) shows 3D-MCF-7 cells
are surrounded by the F-actin stress fibers surrounding the
surfaces, organizing the leading edges of filopodia and
lamellipodia, and lack stress fiber bundles; and figure 7(b)
shows that 3D-MCF-7 cells contain the microtubules that
are radiated by the cytoplasm. From figure 7(b), it is also
observed that cells undergo mitosis and show well-organized
spindle microtubule architectures. The results indicated that
rotary culture did not change the distribution of 3D-MCF-7
cell cytoskeleton.
3.5. Rotary culture activated the ERK1/2 MAPK pathway
Mitogen-activated protein kinases (MAPKs) regulate most
of the cellular processes, including proliferation, death
and differentiation signaling cascades. Extracellular signal-
regulated protein kinase (ERK), c-jun N-terminal kinase (JNK)
and p38 are members of the MAPK family. In this study,
three proteins and their active forms (phosphorylation) were
detected in both rotating and control groups with the results
shown in figure 8. Although there was no notable change
in the amount of ERK1/2 protein in both groups, rotary
culture increases the phosphorylation of ERK1/2 (ERK1/2-
P) compared to the control group. The amounts of JNK,
phosphorylation of JNK (JNK-P) and p38 show insignificant
change in both groups. Phosphorylation of p38 was not
detected in both groups. The results indicated that rotary
culture induced the ERK1/2-MAPK pathway, but did not
activate the JNK or p38 MAPK pathway.
(c)
(b-2)
Biomed. Mater. 7 (2012) 015003 H Zheng et al

(a)

(b)

Figure 7. Comparison of the cytoskeleton distribution of 3D-MCF-7 cells in the rotary culture environment and static condition:
(a) fluorescence images showing the distribution of microfilaments (green) and nuclei (blue), the arrows point to the F-actin stress fibers, and
(b) fluorescence images showing the distribution of microtubules (red) and nuclei (blue), the green arrows point to the microtubule
framework in interphase cells and the white arrows point to the central spindle in dividing phase cells.

4. Discussion

3D tissue culture models play an important role in

Figure 8. Comparison of the protein expression of the MAPK
pathway in 3D-MCF-7 cells in the rotary culture environment and
static condition.
tumor biology by providing insights into cancer biology
[6, 33]. However, in the conventional static 3D culture systems,
the diffusion of nutrient components and metabolic waste is
limited. With the rotation of the RCCS, cells are maintained
in movement, thus promoting the transport of nutrients and
metabolite wastes. As such, the RCSS provides a preferable
environment for cell growth in three-dimensional models
[25, 33, 34]. It is also noted that the RCCS exposes cells
to a SMG environment, thus changing the cell perception of
gravitational direction. Previous studies have demonstrated
that the exposure of cells to microgravity could suppress
cell proliferation, block cell cycle and increase apoptosis
[35–38]. In this study, 3D tissue-like spheroids were
constructed by encapsulating the breast cancer MCF-7 cells
in the CA hydrogel and then cultured in a RCCS. With this
model, we investigated the MCF-7 cell proliferation with
the results demonstrating that rotary culture can enhance the

7
Biomed. Mater. 7 (2012) 015003
MCF-7 cell proliferation in a 3D model via activation of the
MAPK pathway.
The carrier used in this study is hydrogel made
from alginate and collagen. Sodium alginate comprises
mannuronic and guluronic sugars and can be gelled using
ionic cross-linking with divalent cations such as calcium [19].
Previous studies have demonstrated that calcium cross-linked
alginate hydrogels are non-toxic and biodegradable for use
in various biomedical applications, including encapsulating
living cells, wound healing and drug delivery [16–19]. In
cancer research, tumor–matrix interactions are considered
important to the tumor progression [39]. Among the most
common ECM materials, collagen has been used to provide
a microenvironment to study mammary and prostate cancer
progression [12]. In our study, to create the in vivo-mimic
microenvironments, alginate hydrogels were made with the
addition of collagen I. In order to identify mechanically stable
constructs for cellular culture, the mechanical properties of
hydrogel with varying ratios of alginate to collagen were
examined with a rheometer. The results show that by increasing
collagen to a certain level (with a collagen-to-alginate ratio
of 30:1), the CA hydrogel achieves the mechanical stability
suitable for cell culture. On this basis, we chose the hydrogel
of 30:1 collagen/alginate for the cell encapsulation. Further
experiments on the hydrogel encapsulating cells show that
the porous structure of the CA hydrogel is able to enhance
the transport of nutrients and metabolic products, thereby
providing a preferable environment for 3D cell growth.
Microgravity has been recognized as an important factor
for cell culture. It was found that cells exposed to the outer
space microgravity environment tended to aggregate, thus
promoting tissue reconstruction [21]. Various cell types have
been successfully cultured in the SMG environment to form
various tissue-like constructs [21, 25, 40–42]. The influence
of microgravity on the cellular events such as cell cycle,
proliferation and differentiation has been drawing considerable
attention from researchers [26, 35–37, 43–45]. In this study,
the influence of SMG on 3D-MCF-7 cells was examined and
evaluated. In order to prevent interference of culture serum,
1% FBS was used for cell culture in both rotating and control
groups. MCF-7 cell spheroids in rotary culture proliferated
more remarkably than those in static culture, which was
supported by various cell proliferation tests. Firstly, the cell
proliferation was evaluated by the MTT assay. The results
show that the viability of MCF-7 cells cultured in the RCCS
is higher than that of the control group. Secondly, the results
from the cell cycle analysis show that the per cent of the
cells in S and G2/M phase after 48 h of rotating culture is
significantly increased compared to the control group. Finally,
the expression of PCNA and cyclin D1 was examined. PCNA
is an antigen expressed by the nuclei of cells during the
DNA synthesis phase of the cell cycle [46]. Cyclin D1 is a
promoter of the progression of the G1-S phase and, as such, it
plays an important role in cell proliferation, including tumor
proliferation [47]. Consistent with the results of MTT and
cell cycle, PCNA and cyclin D1 expression in the RCCS is
increased compared to the static culture (figure 6(c)). The
examination of the cytoskeleton distribution on the 3D MCF-
7 cells in the RCCS shows its similarity to that in the static
8
H Zheng et al
culture. Taken together, our results show that the RCCS is able
to provide an improved environment over the static culture
condition for MCF-7 cells to grow three-dimensionally. This
would represent a significant benefit to research in the areas of
cancer biology and tissue engineering.
Activation of ERK1/2 is associated with cell survival and
strongly implicated in cell growth, malignant transformation
and drug resistance as a driver of the malignant phenotype
and potential therapeutic target for breast and other cancers
[2], whereas both JNK and p38 are associated with apoptosis
[48]. Previous study showed that SMG activated the ERK1/2-
MAPK pathways in fibroblasts cultured on microgrooved
surface topography [49]. In this study, we examined the
MAPKs, which regulate most of the cellular processes such
as proliferation, death and differentiation signaling cascades.
The results from the western blot assay showed that the RCCS
culture could induce ERK1/2 phosphorylation, but did not
affect the total amount of ERK and did not activate JNK or
p38 MAPK. This suggests that the rotation culture promotes
the proliferation of 3D MCF-7 cells primarily through the
ERK1/2-mediated pathway.
In summary, this paper presents a new 3D carrier, i.e.
the CA hydrogel, as applied to the study on the proliferation
of MCF-7 cells cultured in the RCCS. The results provide
evidence that the RCCS can enhance the proliferation of
MCF-7 cells through the MAPK pathway. Also, the results
suggest that the 3D CA hydrogel not only provides a better
environment for 3D cell growth, but also allows for seeking
insight into the 3D breast cancer biology and developing new
therapies and treatments for breast cancer.
Acknowledgments
This work was supported by the 863 Program In China
(2006AA7035030E), National Natural Science Foundation of
China (NSFC) (50903024), and the Opening Foundation of
the State Key Laboratory of Space Medicine Fundamentals
and Applications through the Chinese Astronaut Research and
Training Center (SFA10K02).
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