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

 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

/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

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

/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

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

/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.

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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).

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