Arbitrary Microstructure Fabrication Embedding Yeast Cells

Patterned by Dielectrophoresis
Tao Yue, Student Member, IEEE, Masahiro Nakajima, Member, IEEE, Masaru Kojima, Member, IEEE,
and Toshio Fukuda, Fellow, IEEE
Department of Micro-Nano Systems Engineering, Nagoya University
Furo-cho, Chikusa-ku, Nagoya, 464-8603 JAPAN

Abstract: and nanowires [5]. Consequently, DEP force is widely used

Constructing different patterns of cells and immobilizing in cell sorting and separation [6].
these cells inside certain structures are very important There are different kinds of cell immobilization methods,
issues for artificial tissue engineering. In this paper, we such as aspiration, pressure of solution and fluidic structure
present methods of forming line pattern of yeast cells by [7]. The advantage of aspiration is the fixing force is large,
dielectrophoresis (DE P) and immobilizing patterned while the disadvantage is damaging the cells [8]. On-chip
cells by photo-crosslinkable resin. Arbitrary shaped fabrication based on photo-crosslinkable resin via UV
microstructure which contains patterned yeast cells is illumination is a creative way for immobilizing cells. There
on-chip fabricated. In order to applying DE P force for are several advantages such as high-speed, low-cost and
forming cell pattern, several novel microelectrodes are arbitrary shaped [9], [ 10].
fabricated by Indium Tin Oxides (ITO) which are coated
Yeast cells
on the glass. The two kinds of DE P responses of yeast Photo·crosslinkable

cell (W303) and the precise experimental parameters of ITO electrodes -=���=::::§:§��� solution

them are confirmed. Based on negative DE P Glass substrate

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phenomenon, cell traps generated by microelectrode are
Microstructure
demonstrated. Besides, the line pattern of yeast cells is
formed. The experimental results show that the cell line
pattern which contains hundreds of yeast cells is formed
by DE P within 1 second. The on-chip fabrication for
arbitrary shapes of microstructures based on Poly
t -;
Microstructures fabricated
,,/ Pattemedyeastcellsinsolution

E thylene Glycol Diacrylate (PEG-DA) is reported. With by PEG-DA ' - - - - - - - - - - - - - - - -,

[00000061 .. : Movablemicroslruclures '
the cell patterning by DE P and immobilizing by on-chip , and further assembly ,
Microstructures with cells ,________________ ,
fabrication, microstructure which contains 3 lines of
Fig.! A schematic drawing of the cell assembly method
yeast cell inside is fabricated in the microfluidic channel,
inside PEG-DA and NaCI solution.
We report the methods of forming line pattern of yeast
cells by DEP and immobilizing by photo-crosslinkable resin.
1. INTRODUCTION
The whole research procedure is shown in Fig. 1. Several
novel microelectrodes are fabricated by Indium Tin Oxides
Currently the research about large quantities cells
(ITO). There are two kinds of DEP responses of yeast cell
assembly is seriously regarded, since it can provide high
(W303) which is positive DEP and negative DEP. The
efficiency methods for artificial tissue engineering. For
precise experimental parameters of them are confirmed.
artificial tissue, important issues are how to form different
With the expectation of applying negative DEP
pattern of cells and how to immobilize these cells inside
phenomenon, matrix cell traps generated by microelectrode
certain structures [ 1].
are fabricated. Line pattern of yeast cells which contain
There are many kinds of cell patterning methods, such
hundreds of yeast cells is formed by these traps within 1
surface adhesion, surface pattern and dielectrophoresis. The
second.
surface adhesion is no damage to cells but the position is not
We also present the on-chip fabrication method for
flexible [2] . The cell traps made of microwells are applied to
microstructures with the expectation to apply for single cell
trapping single cells and cell culture [3]. Compare with other
analysis system. Via the microscope, the patterned UV-ray
methods for forming cell patterns such as aspiration and
is illuminated through the mask to the photo-crosslinkable
surface adhesion, dielectrophoresis (DEP) is easier to
resin which is injected into the microfluidic device made of
control and it is non-contact which means less damage to
Polydimethylsiloxane (PDMS). The photo-crosslinkable
cells [4]. By DEP force, it is possible to manipulate particles
resin, which is Poly Ethylene Glycol Diacrylate (PEG-DA),
with great selectivity. It is able to allow the separation of
has low toxicity [ 1 1]. This resin is polymerized and
cells or the orientation and manipulation of nanoparticles
microstructures with arbitrary shape are fabricated at desired

978-1-4577-1362-0/11/$26.00 ©2011 IEEE - 198 -

place inside the channel of microfluidic device. There is no suitable for being fabricated to micro scale structures. The
need to prefabricate microstructures or to inject the micro glass which is coated by thin ITO is used for fabricating
objects into the channel from outside. It is possible to microelectrode. The thickness of ITO layer is 150 nm . The
fabricate the required microstructures at the desired places. fabrication procedure for ITO microelectrodes is shown in
The fabricated microstructures can be movable on the Fig. 3.
surface of PDMS, so the microstructures can be assembled
to 3D structure and applied to the tissue engineering. Based Positive Dielectrophoresis Negative Dielectrophoresis
on the cell patterning by DEP and immobilizing by on-chip
fabrication, immovable microstructure which contains 3 .-/
",,/

lines of yeast cell inside is fabricated in the microfluidic + .

:
�
•

�
.
·

. .
·

channel, inside PEG-DA and NaCI solution. . + - .· +

....
2. DIELECTROPHORESIS BASED ON MICROELECTRODE Direction of Direction of
movement movement

2. 1 Dielectrophoresis
(a) .,. AC (b) .,. AC
DEP is a phenomenon in which a force is exerted on a
dielectric particle when it is inside a non-uniform electric Fig. 2 The principle of Dielectrophoresis (DEP)
field. This force does not require the particle to be charged.
All particles exhibit dielectrophoretic activity in the
presence of electric fields. However, the strength of the
force depends strongly on the electrical properties of
medium and particles, on the shape and size of the particles,
and on the frequency of the electric field [ 12] . There are two
DEP responses which are positive DEP (p-DEP) and
negative DEP (n-DEP). The basic principle of DEP theory is
shown in Fig. 2.
For a spherical particle of radius r suspended in a medium

of permittivity [;m' the dielectrophoretic force is given by

the expression:

FDEP = 27r[;mr3 Re[fcM]' V(E;ms) ( 1)

Where E rms is the root mean square value of electric field.

Fig. 3 Fabrication procedure of ITO microelectrode and PDMS
Re[fCM] means a real part of the fCM' which can be microfluidic chip

represented as follows:
•

- m
•

fCM= [;p' [; ' (2)

[;p + 2[;m
Where [;' is the complex permittivity which is calculated
'
by [; = [; - j () / OJ. () is the conductivity and OJ is the

frequency of electric field. Subscripts p and m mean particle

Re[fcM] > 0
and the medium, respectively. Therefore,

means that particle shows positive DEP response while

1
Electric field

Re[fcM] < 0 means negative DEP response [ 13].

Fig. 4 The image of one microelectrode made of ITO and the schematic of
wire connection

2.2 Microelectrode made of ITO

In order to apply DEP to manipulate cell, it is needed to Fabrication method is based on photolithography. First,
develop special electrodes of which the size is 25x25 mm size ITO glass is prepared. Second, the
approximately as much as the size of cells. This electrode is photoresist AZ is coated on the surface of ITO layer. Third,
very small that we call it microelectrode. Microelectrode is with photolithography instrument, the AZ coated ITO glass
the most important element for cell manipulation by DEP. is exposed by the patterned UV which is as same as the
For the conductive material, we choose ITO because it is designed microelectrode. Finally, with the patterned AZ

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coated layer, ITO microelectrode is fabricated by chemical Therefore, it is possible to use this structure to trap yeast
etching. The image of one microelectrode is shown in Fig. 4. cells and then control the position of yeast cell.

p·DEP n·DEP
2.3 Microfluidic chip made of PDMS
The DEP force should be generated inside solution which
means the microtluidic channel is needed. Microtluidic chip
made of polydimethylsiloxane (PDMS) is a key thing for Yeast cells
/
many microtluidic applications. The fabrication procedure
for PDMS microfluidic chips is shown in Fig. 3.
Edges of ITO
Fabrication method is also based on photolithography. electrodes ""'�T---t---�-I-----J
First, the mold of the microfluidic channel is made by
photoresist SU-8. Second, the SU-8 mold is covered by �
Fig. 5 Experiment results of positive dielectrophoresis (p·DEP) and
PDMS liquid and it will solidify after 24 hours. Third, the
negative dielectrophoresis (n·DEP)
solidified PDMS with the channel is detached from the mold A
and then covered on the glass. In order to put the tube and
inject the resin, holes are made on the ends of the channel.
Finally, the silicon tube is put in the hole and the solution is
injected to the channel by the negative pressure.

3. LINE PATTERN OF YEAST CELL FORMED BY DE P

3. 1 Two different kinds of D E P responses of yeast cell

Based on the principle of DEP, there are two DEP
responses of yeast cell which is p-DEP and n-DEP.
Traps ITO electrodes
Alternating Current (Ae) and NaCI water solution are used AC
for generating DEP force. By adjusting the frequency of AC Fig.6 The numerical simulation of electric field of the microelectode

and concentration of NaCI solution (medium conductivity),

these two DEP responses will be generated [ 12]. The
reasons of using AC are as following. First, operating at
high frequencies eliminates any electrophoretic movement
of the cell due to its charged membrane. Second, such
operation eliminates electrochemical reactions at the
electrode-electrolyte interfaces, preventing electrode
.2aSE-03 .368514 .73674 1.105 1.473

corrosion and gas formation [ 14]. DEP-2

.184401 .552627 .920853 1.289 1.657

In order to confirm the DEP responses of yeast cell and

Fig.7 The numerical simulation of the electric field distribution over the
precise parameters of p-DEP and n-DEP, an experiment is microelectode at the cross section, A·A' of Fig. 6
performed and the result is shown in Fig. 5. The
microelectrode we used is the one described in Section 2.2.
For generating p-DEP, concentration of NaCI solution is 90
mg/L, frequency of AC is lK Hz. For generation n-DEP,
concentration of NaCl solution is 200 mg/L, frequency of
AC is 500K Hz. In both cases, Vpp of AC is 8 V.

3.2 Cell traps generated by negative DEP

With the consideration of manipulating yeast cells by
DEP, usually the n-DEP is used. Special structures of
electrode are applied to trap cells in desired positions based
AC
on n-DEP [ 15- 17]. One kind of cell trap is designed and
Fig.8 The image of line circles made of ITO microelectrode and one line of
numerical simulated. The schematic of the design is shown
5 yeast cells
in Fig. 6. The simulation of the electric field is shown in Fig.
[Link] cell traps are generated in the circles which are the The fabrication procedure of this microelectrode is
holes in the electrode. By the simulation result of electric mentioned in Section 2.2. With the parameters for
field, the lowest density positions of electric field are the generation n-DEP, the experiment of trapping yeast cells is
center of the circles. The particles inside this electric field performed. One line of 5 yeast cells is formed. As shown in
will trapped in the circles when the n-DEP is generated. Fig. 8, yeast cell is trapped inside the circle.

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3.3 Line pattern of yeast cells exposing UV on samples. For the motion to change the
With the expectation of applying n-DEP to form more observation range and manipulate the objects, X-Y stage and
complex pattern of yeast cells, matrix cell traps which made height of the objective lens (Z-axis) are controlled by
of several lines electrodes are fabricated, as shown in Fig. 9. manual. The oil immersion objective (UPLFLN 100XOI2,
The size of trap and electrode is about 11 flm and 30 flm, Olympus) is used. The magnification is 100 and N.A. was
which is suitable for the size of yeast cell. Based on the 0.6� 1.3. UV is illuminated by the mercury lamp
result of Section 3.2, yeast cells will be trapped in the circle. (USH- 103tems) using DC mOL, Olympus) controlling the
If the amount of cell is adjusted well, several lines of cell shutter (BSH-RIX, Sigmakoki). The microfluidic device is
will formed by n-DEP. put on the stage. The experiment and observation are
By this novel designed micro electrode, the cell line performed using the CCD camera (XC-555, Sony).
patterns which contain hundreds of yeast cells are formed as
shown in Fig. 10. Besides, the experimental results 4.2 On-chip fabrication method of arbitrary
demonstrate that the cell line pattern is formed within 1 microstructures
second which shows the potential to be used in high-speed As shown in Fig. 1 1, the mask made of PET
cell manipulation for tissue engineering. (polyethylene terephthalate) is set in front of the shutter on
the optical axis. The arbitrary shape patterns are printed on
the mask. UV is illuminated through this mask from bottom
of the objective lens into the PEG-DA (molecular weight
200) which is one kind of photo-crosslinkable resin inside
the channel of PDMS (SILPOT 184 WIC, Dow Coming
Toray) microfluidic chip. UV which exposes into
microscope is patterned by the masks. Then the
photo-crosslinkable resin is polymerized and the arbitrary
shapes of microstructures will be fabricated inside the
solution [ 18]. The fabrication time usually is within I
second.

Photo-crosslinkable resin Microstructure

�
Glass substrate

Objective lens
Fig.9 The matrix DEP traps made of 5 line ITO microelectrodes. Diameter
of the traps is 11 �m and width of each electrodes is 30 �m.

Microstructure

Channel -{
POMS � :�zL
}
·'
h
''''
l
P

Fig. 11 Schematic drawings of the on-chip fabrication method of

microstructures
[Link] The line pattern of yeast cells formed by n-DEP based on ITO
microelectrode
The position of the microstructure is confirmed by the
4. ON-CHIP FABRICA nON METHOD FOR ARBITRARY stage. When the surface of bottom in the channel is glass,
MICROSTRUCTURES the fabricated microstructure adheres on the glass surface.
When the surface of bottom in the channel is the PDMS, the
4. 1 Equipment of on-chip fabrication system fabricated microstructure is able to move in the
The on-chip fabrication method for arbitrary shapes of non-polymerized resin freely without adhering. Because the
microstructures is shown in Fig. II. Optical microscope oxygen layer which exists near the PDMS surface inhibits
(IX-71, Olympus) is used for observing samples and the polymerization of photo-crosslinkable resin [ 19]. PDMS

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has the permeability to air and it adhere one thin layer of ITO electrode Microfluidic chip channel
oxygen. This oxygen molecule inhibits the polymerization. -------------- � ----------
I Outiet Inlet I
Therefore, the polymerization does not occur near the
s= ¢=?
PDMS surface and the fabricated microstructure moves in 1 ___ =��====���====
the channel freely.
The fabricated microstructures are shown in Fig. 12. They Patterned yeast cells

are fabricated by illuminating the UV-ray for 0.2 second via

the oil immersion objective lens of xl 00 (UPLFLN uv
100X0I2, Olympus).
Fig. 13 The experimental setup of fabricating microstructure embedding
patterned yeast cells.

Fig. 14 The microstructure embedding patterned yeast cells. It contains 3

Fig. 12 Arbitrary shapes of on-chip fabricated microstructures
lines of yeast cell inside.

5. FABRICAnON OF MICROSTRUCTURE E MBEDDING

the line patterns of yeast cells are formed, donuts shaped
PATTERNED YEAST CELLS
microstructure is fabricated at the same position. This
microstructure contains 3 lines of yeast cells,as shown in
5. 1 Experimental setup
Fig. 14. The inner and outer diameter of the donuts shape is
Based on the cell patterning method which use DEP and
35 /lm and 100 /lm. Yeast cells cakes because of the
on-chip fabrication method for microstructure, the
PEG-DA inside the solution.
fabrication of microstructure embedding patterned yeast
In this microstructure, the number of yeast cells is almost
cells is demonstrated.
100, and they are formed into the pattern of 3 lines. The
The experimental setup is as following. First, ITO
pattern was generated in 10 seconds, and the microstructure
microelectrode as shown in Fig. 9 and PDMS microfluidic
was fabricated in 0.2 second. Therefore, it is possible to
chip as shown in Fig. 3 is prepared. The ITO microelectrode
fabricate this kind of microstructures very quickly and
is covered by PDMS chip. Second, 50% PEG-DA
immobilize large amount of yeast cells inside them. It shows
(molecular weight 700) and 200 mg/L NaCI water solution
the great potential to be used in high-throughput cell
is prepared. The reason why we use PEG-DA (700) is that it
patterning and immobilization for tissue engineering.
is able to dissolve inside the water. Then the yeast cells
(W303) are mixed into the solution. Third, the solution is
6. CONCLUSION
injected into the microfluidic channel. The AC power supply
is used for generating DEP force. Frequency of AC is 500K
We present methods of forming line pattern of yeast cells
Hz, and Vpp of AC is 8 V. Fourth, after the cell pattern is
by negative DEP and immobilizing patterned cells by
formed, the objective lens is adjusted to the position of the
photo-crosslinkable resin which is PEG-DA. The
patterned cells. Then UV is exposed for 0.2s to fabricating
microstructure which contains patterned yeast cells is
the microstructure at the same position as the patterned cells.
fabricated. Several novel microelectrodes are fabricated by
The whole procedure is shown in Fig. 13.
ITO which is coated on the glass. The positive and negative
DEP response of yeast cell (W303) is observed and the
5.2 Experimental results
precise experimental parameters of them are confirmed. Cell
This time the donuts shaped mask is used. Therefore, after
traps based on negative DEP are generated by

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