MICROFLUIDIC CELL STIMULATOR USING BEAD IMPACT
Young-Hun Kim, Tae-Jin Kim, Hyung-Joon Kim, Min-Suk Park and Hyo-Il Jung
School of mechanical engineering, Yonsei University, Seoul, Republic of Korea
Keywords: Mechanical stimulation, Micro device, Rolling bead, CPAE, PDMS.
Abstract: Recently many researchers are focused on cell stimulation regarding observation of cells to specific
stimulation factors. We introduce new mechanical stimulation method using micro beads without any
chemical reagents. CPAE (calf pulmonary artery endothelial cell) were cultured in PDMS
(polydimethylsiloxane) microfluidic device. After starvation process, sterilized 10μm glass beads were
rolled by only gravitational force for 10 minute. To find optimal stimulation time, 16 devices were made by
PDMS and each device was slanted every hour. Results show that cell exposed under micro bead
stimulation perform at a higher growth rate than normal conditions and 1 hour stimulation time represents
more effective than other stimulation times. This new cell stimulation method will be able to help make
artificial organ such as blood vessels in the future.
1 INTRODUCTION
Studies about observation of cell behaviour are
performed to explore factors which are related with
inhibition and promotion of cell growth. It is true
that cells in human and animal are exposed under
various stimulation such as mechanical (Matteucci,
2007), chemical (Nakashima, 2005), and electrical
stimulation (Mattei, 2004). These studies were tried
to find alternative stimulation of cell growth, thus
could contribute tissue regeneration. However the
effect of mechanical stimulation is not to be
accomplished and possibility of stimulation is still
opened.
Miniaturized bioreactor for cell stimulation was
developed by soft lithography and PDMS
(
polydimethylsiloxane) molding technique. It is
considered inexpensive and time saving method and
makes disposable device and requires small culture
media and other reagents. Moreover PDMS has bio-
compatible and gas permeability. By using PDMS
device, we could fabricate micro cell stimulation
system with a small amount of culture media and
other reagents (Kim et al., 2008). Various methods
for physical cell stimulation such as fluidic shear
stress (Brown., 2008), pneumatic pressure (Sim,
2006), microfluidic motion (Pioletti, 2003), and etc.
have developed. In light of this perspective; we
expect that micro-beads can be one of effective
sources for physical stimulation to improve cell
growth.
Now, we demonstrate a novle stimulation
method using micro-bead impact and microfluidic
device. The mechano-stimulation by bead impact
will be one of the efficient stimulation methods.
2 MATERIAL AND METHOD
2.1 Fabrication of Stimulation Device
Various experiments were conducted in order to find
an optimal condition for cell culture in microfludic
culture chambers, and the first step is to determine
the appropriate method to fabricate devices. Our
choice was to use soft lithography and PDMS to
easily acquire the required microfluidic devices.
A set of single straight channels were created on
a Silicon wafer using negative photoresist Su-8
mold. After treating a bare silicon wafer with
acetone, methanol and DI (deionized) water,
respectively, Su-8 2050 (MicroChem, MA, USA)
negative photoresist was spin-coated at 1,300 rpm
for 30 seconds to acquire a channel height of 100
μm. After the soft baking process, the wafer was
exposed under UV light followed by additional heat
treatment for post exposure baking process. The
wafer was etched using Su-8 developer and was
426
Kim Y., Kim T., Kim H., Park M. and Jung H. (2009).
MICROFLUIDIC CELL STIMULATOR USING BEAD IMPACT.
In Proceedings of the International Conference on Biomedical Electronics and Devices, pages 426-429
DOI: 10.5220/0001780204260429
Copyright
c
SciTePress
rinsed using iso-propyl alcohol (Figure 1).
Figure 1: Soft lithography fabrication process using Su-8
photoresist.
2.2 Cell Culture
CPAE (calf pulmonary artery endothelial cell) was
selected to investigate the change in growth rate
when exposed to micro bead impact. These cells are
thought to be exposed to blood cells impact in a
blood vessel. It can be thus, mimicry of blood cells
impact that micro beads collide against the surface
of CPAE. Sterilization was performed under UV for
24 hour 70% ethanol. The cells were seeded into the
microfluidic chamber with a concentration of
1.65x10
6
/ml and were incubated overnight. Before
experiments, starvation process was conducted with
0.5% FBS RPMI media for 24 hours to fix cell phase
in G1.
2.3 Experimental Setup
In previous work, two types of cell lines, HeLa cell
and MC3T3 cell, were selected to investigate the
changes in growth rate when exposed to micro-bead
impact. The cells were seeded into the microfluidic
chamber with a concentration of 5 x 10
5
cells/ml and
were incubated overnight. Fresh DMEM was
supplied in HeLa cell cultured chambers and aMEM
in MC3T3 cell cultured chambers every 12 h. Since
the concentration of 11μm micro-beads in culture
medium is 10
6
/ml, and the flow rate is 3μl/min, the
number of 11μm micro-beads passing through a
single straight cell culture chamber per minute is
calculated as 3 x 10
3
beads/min (Figure. 2)
However, this simulation system was designed
except important aspects. It had two mechanical
stimulus factors, flow and bead impact and it is not
considered about cell cycle which the series of
events that take place in a cell leading to its
replication. Therefore we developed idea to
minimize other stimulus sours and maximize bead
impact to cells. Also we performed experiment with
considering cell cycle to find optimal stimulation
time and to get effect of the number of micro-bead
rolling in cell growth.
Sixteen micro devices have been fabricated
because cell cycle is about 16 hour and each device
has 10 cell culture chamber which dimensions are
height = 100μm, width = 40μm, length = 80μm
(Figure 3). There were two inlets, one for cell
seeding and media and other for beads. The
concentration of 10 μm glass beads is 1.9x10
5
/ml.
Before tilting device, micro beads were gathered on
inlet for bead. Slating process was performed for 10
minutes in incubator.
Figure 2: Photograph and schematic diagram of previous
work about cell stimulation.
Figure 3: Schematic diagram and photograph of device.
MICROFLUIDIC CELL STIMULATOR USING BEAD IMPACT
427
3 RESULT
The micro-bead and cell culture medium mixture
was introduced into the microfluidic chamber using
syringe pump infusion mode, and deflections in the
micro-bead pathway were observed as they flowed
over or by the adhered cells. This motion indicates
that micro-beads flow along the adhered cell’s
boundary layer and the outer membrane of the cells
are stimulated through this manner. The cell
population of HeLa and MC3T3 cells under different
micro-bead stimulation was counted through
microscopic observation at a 12-h interval, and the
proliferation rate was then achieved by dividing the
cell number in each time interval by the initial
population. The cell increase rate of HeLa cells in
the experimental chamber with 11-lm micro-bead
stimulation performed the highest growth rate,
whereas 2μm micro-bead stimulation performed the
lowest growth rate. For the case of MC3T3 cells, the
cell increase rate of the 2μm micro-bead stimulation
device was significantly higher than that of the 11-
lm micro-bead stimulation device, while the control
group performed the lowest stimulation rate (Fig. 4).
Cell growth was measured in 16 type device:
control device and other related with each
stimulation time. The cell population of HeLa and
MC3T3 cells in the control and experimental
chambers was observed through an inverted optical
microscope (Olympus, Japan) and compared after 16
hours. All fluids were supplied by capillary force to
remove effect of flow. Figure 5 shows increase of
CPAE cell according to the stimulation time. The
proliferation rate (Figure 6) was then achieved by
dividing the cell number in each time interval by the
initial population.
As mentioned above, cells are divided by cell
cycle which consists of G1, S, G2, and M phase.
First, G1 phase is marked by synthesis of various
enzymes that are required in S phase, mainly those
needed for DNA replication. Next, S phase is related
with DNA synthesis. Third, G2 phase significant
protein synthesis occurs which are required during
the process of mitosis. Finally in M phase the cell is
divided by two cells.
According to figure 6, we could conclude that
micro bead stimulation was more effective at 1 hour
data than other stimulation time. We analyzed that 1
hour stimulation time was involved in G1 phase
because cells were fixed by starvation process. G1
phase takes part in synthesis of diverse proteins
about DNA synthesis. Therefore mechanical
stimulation by rolling micro bead plays an important
role to proliferation of cells. Following this data, we
performed other experiment to verify effects of the
number of rolling micro-beads. Figure 7 shows that
more frequent micro-beads stimulation could be
better effect in cell growth.
Figure 4: Cell number increase comparison of control
group. : (a) HeLa cells and (b) MC3T3 cells.
Figure 5: Photograph of CPAE cell in micro chamber : (a)
control device, (b) device at 1 hour stimulation time,(c)
device at 15 hour stimulation time.
Figure 6: Comparison of growth rate of CPAE cells under
different stimulation time: blue bar for data at 0 hour and
yellow bar for data at 16 hour.
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428
Figure 7: Comparison of growth rates of CPAE cells under
different the number of beads rolling.
4 CONCLUSIONS
A new method for stimulating cells has been
introduced. Previous our work was conducted with
two types of polystyrene micro-beads: 2 and 11μm
micro-beads. The cell population increase rate was
the highest when stimulated by 11 μm beads in the
case of HeLa cells and 2 μm beads in the case of
MC3T3 cells. From the comparison between control
device and experimental device, it can be seen that
cells in the experimental group performs at a higher
growth rate. This suggests that the growth rate is
accelerated when stimulated by micro-beads. We
designed new device to maximize micro bead
stimulation and minimize other factors: beads were
rolled by only gravitational force. Thus, we get data
about more effective simulation time, 1 hour which
is involved in G1 phase and cells exposed under
micro bead impact represent better proliferation.
Moreover, the number of bead impact is one of the
important factors in cell stimulation and more
frequent impacts show better effects to the cells.
When our new devices which were designed to
maximize beads impact was compared with previous
our device, cell proliferation data by only beads
impact represent similar increase rate (about
10%~40%). Therefore we could conclude that
micro-beads stimulation can be one of the effective
physical stimulation to enhance cell proliferation.
Now, we are conducting additional experiments
to convincing our experiments including cell
viability test and we will perform other analyzing
test to compare with other research data such as
protein analysis.
ACKNOWLEDGEMENTS
This work was supported by National Core Research
Center (NCRC) for Nanomedical Technology of the
Korea Science & Engineering Foundation (Grant no.
R15-2004-024-01001-0), Seoul Research &
Business Development (R&BD Program, 11128)
and Korea Research Foundation Grant funded by the
Korean Government (MOEHRD) (KRF-2007-313-
D00073).
REFERENCES
Matteucci M, 2007, Preliminary Study of
Micromechanical Stress delivery for Cell Biology
studies. Microelectron, Eng 84:1729–173
Nakashima Y, 2005, Microfluidic device for axonal
elongation control, Transducers, Seoul, Korea, 5–9
June
Mattei MD, 2004, Effects of Physical Stimulation with
Electromagnetic Field and Insulin growth factor-I
treatment on proteoglycan synthesis of bovine
articular cartilage. Osteoarthritis Cartilage 12:793–
800
Tae-Jin Kim, Su-Jin Kim, Hyo-Il Jung, 2008, Physical
stimulation of mammalian cells using micro-bead
impact within a microfluidic environment to enhance
growth rate, Microfluid Nanofluid on-line published
Brown TD, 2000, Techniques for mechanical stimulation
of cell in vitro: a review, J Biomech, 33: 3-14
Sim WY, 2006, A pneumatic micro cell simulator for the
differentiation of human mesenchymal stem cell
(hMSCs), MicroTAS, Tokyo, Japan,5-9
Pioletti DP, 2003, Effect of micromechanical stimulations
on osteoblast: development of a device stimulating the
mechanical situation at the bone-implant interface, J
Biomech 36: 131-135
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