Design of Scaffold with Array of Micro Projections
to Trace Intra- and Inter-cellular Behavior
Yusuke TAKAHASHI, Kenta SUGIMOTO, Haruka HINO, Tomoaki KATANO, Shigehiro HASHIMOTO
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACT

micro location of the surface.

The transparent micro-patterned scaffold has been designed toobserve the interaction between cultured cells in vitro. The
scaffold consists of a thin cover sheet and arrangement of
micro-pillars. The pillars of polydimethylsiloxane were madeby the photolithography technique. Each pillar has thecylindrical shape (0.007 mm diameter, 0.002 mm height). The
distance between adjacent pillars is 0.003 mm. The thicknessof the base sheet of the pillars is 0.02 mm. The sheet withpillars is turned upside down, and used for the scaffold of cell
culture. The pillars simultaneously play a roll of the position
marker. L929 (fibroblast connective tissue of mouse), or
C2C12 (mouse myoblast) was seeded on the sheet at the countersurface to the pillars at the density of 1000 cells/cm2. The
cells were cultured in the medium containing 10% FBS (fetal
bovine serum) and 1% penicillin/ streptomycin. Cells are able
to be traced through the transparent scaffold, but the
deformation of scaffold cannot be detected. The designed
scaffold has a potential to trace the intracellular andintercellular behavior microscopically in vitro.

In the present study, the transparent micro-patterned scaffold
with the micro-pillar array has been designed to observe theinteraction between cultured cells in vitro.

Keywords: Biomedical Engineering, Cell Culture, L929,
C2C12, Micro Pillar, and Photolithography.

Without Photomask
The borosilicate glass (Tempax) disk (50 mm diameter, 1mm
thickness) was used for the base of the mold. The disk wascleaned by the oxygen plasma ashing (100 W, for ten minutes)
in RIE (FA-1, Samco International, Kyoto, Japan).
To
improve affinity between glass and photoresist material, HMDS
(hexamethyldisilazane: Tokyo Chemical Industry Co., Ltd.,Tokyo) was coated on the disk at 3000 rpm for 30 s with a spincoater.

1. INTRODUCTION
Biological tissues actively maintain the morphology by the
intra- and inter-cellular forces. The morphology of each cell is
kept by the interaction between skeletal proteins. Interaction
between cells keeps the shape of the tissue. Interaction
between the extracellular matrix and cells governs thecompliance of the tissue.

2. METHODS
Micro Pattern
The transparent micro-patterned scaffold has been designed toobserve the interaction between cultured cells in vitro (Fig. 1).
The scaffold consists of a thin cover sheet and a micro-pillar
array. The pillars were made of polydimethylsiloxane by thephotolithography technique. Each pillar has the cylindricalshape (0.007 mm diameter, 0.002 mm height). The distance
between adjacent pillars is 0.003 mm. The thickness of thebase sheet of the pillars is 0.02 mm. The sheet with pillars isturned upside down, and used for the scaffold of cell culture.
The pillars simultaneously play a roll of the position marker.
The pattern is drawn in the square area of 0.5 mm × 0.5 mm.

MediumPDMS

Cell

The cell culture technique realizes synthesize the biologicaltissue in vitro. The tissue should have enough strength for theclinical application in regenerative medicine.
Measurement of intra- and inter-cellular forces has been triedby several preparations: using laser, atomic force microscope,and fluorescence technique. At the end of test, scanningelectron microscope, and stain technique are available.
Photolithography technique realizes design of micro
morphology of surface of the scaffold for cell culture [1-9].
The technique can be applied to control compliance of thesurface [7, 8]. The micro morphology can be a marker on theMicropillar
Deformation

Cell

Micropillar

Fig. 1: Model of local deformation (lower) at scaffold sheet
lined with micro-pillar array.

The positive photoresist material of OFPR-800LB (Tokyo Ohka
Kogyo Co., Ltd, Tokyo, Japan) was coated on the disk at 5000
rpm for 30 s with the spin coater. The photoresist was bakedin the oven at 368 K for three minutes.
The pattern for micro-pillars was drawn on the photoresist witha laser drawing system (DDB-201K-KH, Neoark Corporation,
Hachioji, Japan). To control the dimension of the pattern on
the mold with the laser drawing system, the parameters wereselected as follows: the voltage of 2.5 V, the velocity of 0.02
mm/s, the acceleration of 0.5 mm/s2. The pattern was bakedon the heated plate at 368 K for three minutes. The photoresist
was developed with tetra-methyl-ammonium hydroxide(NMD-3, Tokyo Ohka Kogyo Co., Ltd., Kawasaki, Japan) for
five minutes. The pattern was rinsed, and dried by the
spin-dryer.
The dimension of the micro-pillars of the mold was measured
with a laser microscope (VK-X200, Keyence Corporation,
Osaka, Japan). The height along the cross sectional line of
micro pillars was traced.
PDMS (Sylgard 184 Silicone Elastomer Base, Dow Corning
Corp., MI, USA) was mixed with the curing agent (Sylgard 184
Silicone Elastomer Curing Agent, Dow Corning Corp., MI,
USA). The volume ratio of PDMS to curing agent is ten toone. After degassing, 0.3 cm3 of PDMS was poured on themold on the spin coater at 2000 rpm for 30 s, and the ring of
PDMS (outer diameter of 20 mm, inner diameter of 10 mm)
was put on it. PDMS was baked at 373 K for one hour in anoven (DX401, Yamato Scientific Co., Ltd). The baked film of
PDMS was removed from the mold in the acetone by lift off
method (Fig. 2).
The dimension of the micro-pillars at the film was measured
with a laser microscope.
With Photomask
The borosilicate glass (Tempax) disk (50 mm diameter, 1mm
thickness) was used for the base of the mask.
Afterhydrophilization by the oxygen plasma ashing by RIE (FA-1,
Samco International, Kyoto, Japan), titanium was coated on thesurface. To improve affinity between titanium and photoresist
material, HMDS (hexamethyldisilazane: Tokyo Chemical
Industry Co., Ltd., Tokyo) was coated on the disk. The
positive photoresist material of OFPR-800LB (Tokyo Ohka
Kogyo Co., Ltd, Tokyo, Japan) was coated on the disk at 2000
rpm for 30 s with the spin coater.

thickness) was used for the base of the mold. The positive
photoresist material of OFPR-800LB was coated on theborosilicate glass disk. The surface of mask coated withtitanium was mounted on the surface of OFPR-800LB, and thephotoresist was exposed to the UV light through the mask in themask aligner (M-1S, Mikasa Co. Ltd., Japan) for the following
time: 5 s, 8 s, 10 s, 15 s.
The dimension of the micro-pillars of the mold was measured
with a laser microscope.
After degassing, PDMS was poured on the mold on the spincoater, and the ring of PDMS (outer diameter of 20 mm, innerdiameter of 10 mm) was put on it. PDMS was baked at 373 K
for one hour in an oven. The baked film of PDMS was
removed from the mold in the acetone by lift off method (Fig.
2).
The dimension of the micro-pillars at the film was measured
with a laser microscope.
Another ring of PDMS (outer diameter of 20 mm, innerdiameter of 10 mm, and height of 1 mm) was put on the backside, and baked at 373 K for one hour in an oven (Fig. 3).
The culture dish of PDMS was exposed to the oxygen gas at
power of 50 W for one minute in a reactive ion etching system(FA-1, Samco Inc., Kyoto) to be characterized as hydrophilic(oxygen plasma ashing). The dish was preserved in theultrapure water to keep the hydrophilic property of the surface,
before the cell culture.
Cell Culture
Two kinds of cells were used in the test. C2C12 (mouse
myoblast cell line originated with cross-striated muscle of C3Hmouse), L929 (fibroblast connective tissue of C3H mouse).
EMEM (Eagle's Minimum Essential Medium) containing 10%
FBS (decomplemented fetal bovine serum) and 1% penicillin/
streptomycin was used for the culture medium of L929.
D-MEM (Dulbecco’s Modified Eagle’s Medium) containing
10% FBS (decomplemented fetal bovine serum) and 1%
penicillin/ streptomycin was used for the culture medium of
C2C12.
L929 was seeded over the pillars at the density of 1000
cells/cm2 (Fig. 4).

The pattern for micro-pillars was drawn on the mask with alaser drawing system. The photoresist was developed withtetra-methyl-ammonium hydroxide (NMD-3, Tokyo Ohka
Kogyo Co., Ltd., Kawasaki, Japan).
The titanium coating disk was etched with the plasma gas using
RIE-10NR (Samco International, Kyoto, Japan). For etching,
the gas of SF6 (50 cm3/min at 1013 hPa) with Ar (50 cm3/min at
1013 hPa) was applied at 100 W at 4 Pa for five minutes.
OFPR-800LB was removed by acetone, after confirmation ofpattern of etching by the microscope.

Fig. 2: Lift off.

The dimension of the micro-pillars at the mask was measured
with the laser microscope.
The borosilicate glass (Tempax) disk (50 mm diameter, 1mm

Fig. 3: Scaffold sheet lined with micro-pillar array contained on
the ring of PDMS.

C2C12 was seeded on the sheet at the counter surface (backside) to the pillars at the density of 1000 cells/cm2.
The culture dish was kept in the incubator to maintain both thetemperature of 310 K and the carbon dioxide partial pressure of
5 percent. The cells were observed with the phase contrast
microscope (IX71, Olympus, Tokyo) during the cell culture forfive days. The medium was changed every two days.

Micro-pillararray

3. RESULTS
In the case of “without mask”, the tracing of the surface
morphology across the lines of pillars on the mold (Fig. 5)
measured by the laser microscope is exemplified in Fig. 6.
The tracing shows the height of 0.002 mm, the diameter of
0.0078 mm, and interval of 0.0072 mm (diagonal 0.0028 mm).

Fig. 7: Scaffold lined with micro-pillar array (without mask).

In the case of “without mask”, the tracing of the surface
morphology across the lines of pillars on the film (Fig. 7)
measured by the laser microscope is exemplified in Fig. 8.
The tracing shows the height of 0.0024 mm, the diameter of
0.007 mm, and interval of 0.008 mm (diagonal 0.0036 mm).

Fig. 4: L929 cultured on micro-pillar array (left), C2C12
cultured on sheet lined with micro-pillar array (right).

Fig. 5: Mold for micro-pillar array (without
Dimension from left to right is 0.7 mm.

mask).

Fig. 6: Dimension of mold for micro-pillar array (withoutmask).

Fig. 8: Dimension of micro-pillar array (without mask).

Fig. 9: Mask for micro-pillar array.
right is 0.7 mm.

Dimension from left toFig. 10: Dimension of mask for micro-pillar array.

At the mask (Fig. 9), the tracing of the surface morphology
across the lines of pillars on the film measured by the laser
microscope is exemplified in Fig. 10. The tracing shows thediameter of 0.0055 mm, and interval of 0.0094 mm (diagonal
0.005 mm).
In the case of “without mask”, the tracing of the surface
morphology across the lines of pillars on the mold (Fig. 11)
measured by the laser microscope is exemplified in Fig. 12.
Fig. 11 shows the mold with the exposure of 8 s. The tracingshows the height of 0.0033 mm, the diameter of 0.0063 mm,
and interval of 0.0081 mm (diagonal 0.0039 mm) (Fig. 12).
The measurement of the molds shows underexposure (5 s (Figs.
13 &14)) and overexposure (10 s (Figs. 15 & 16) and 15 s).

Fig. 11: Mold for micro-pillar array (8 s with mask).
Dimension from left to right is 0.7 mm.

Fig. 12: Dimension of mold for micro-pillar array (8 s withmask).

Fig. 13: Mold for micro-pillar array (5 s with mask).
Dimension from left to right is 0.7 mm.

Fig. 14: Dimension of mold for micro-pillar array (5 s withmask).

Fig. 15: Mold for micro-pillar array (10 s with mask).
Dimension from left to right is 0.7 mm.

Fig. 16: Dimension of mold for micro-pillar array (10 s withmask).

Fig. 17: Dimension of micro-pillar array (with mask).
Dimension from left to right is 0.7 mm.

Fig. 18: Dimension of micro-pillar array (with mask).

Fig. 21: C2C12 on micro-pillar array, day 1 (24 hours).
Dimension from left to right is 1 mm.

Fig. 19: L929 on micro-pillar array, day 1 (24 hours).
Dimension from left to right is 1 mm.

Fig. 20a: L929 on micro-pillar array, day 2.
left to right is 0.2 mm.

Fig. 22a: C2C12 on micro-pillar array, day 5.
from left to right is 0.4 mm.

Dimension

Fig. 22b: C2C12 on micro-pillar array, day 5.
from left to right is 1 mm.

Dimension

Dimension from

Fig. 17 shows the micro-pillar array made with mask. The
tracing shows the height of 0.0028 mm, the diameter of 0.0041
mm, and interval of 0.010 mm (diagonal 0.0059 mm) (Fig. 18).
The behavior of L929 on the micro-pillar array is exemplified in
Figs. 19 & 20.

Fig. 20b: L929 on micro-pillar array, day 2.
left to right is 1 mm.

Dimension from

The behavior of C2C12 on the micro-pillar array is exemplified
in Figs. 21 & 22. One of the positions of the micro-pillar,
where C2C12 adheres, is shifted on the day 1 (24 hours aftercell seeding).
Myoblast differentiated to myotubes on the sheet in five days
(Fig. 22). Most of longitudinal direction of myotubes orientalong the diagonal direction of the square of array. The closer
direction between micro pillars is diagonal. Several positionsof the micro-pillar are shifted on the day 5.

is available to trace the intracellular and intercellular
deformation in vitro.

6. ACKNOWLEDGMENT
4. DISCUSSION
Adhesion governs the behavior of cells. When the biologicaltissue adheres to the scaffold, the scaffold deforms as the tissue
deforms. The force can be calibrated by the compliance of thescaffold, when the local deformation of the scaffold is traced[10].
In the test of L929, cells are cultured on the pillars. The pillars
of 0.007 mm diameter should be taller than 0.003 mm to be
deformed with the behavior of cells. If the pillars are tall, thecells fall into the valley between the pillars [9]. The cellextends through the slit between the pillars. If the pillar isshort, the compliance of the pillar is too low to makeself-deformation with cells.
In the previous study [6], cells were cultured on the substratewith micro-pillar array. A biological cell adheres, migrates,
rotates, and deforms on the scaffold. These behaviors of celldepend on the morphology of the scaffold. The previous study
show that the orientation of myoblast depends on the height ofthe micro ridges [9]. In the present study, the flat thin sheet
covers the micro pillars, and cells are cultured on the sheet, sothat the cell can adhere on the sheet at any direction regardlessof the position of the micro pillar.
The myotubes make orientation along the diagonal direction of
the sheet of scaffold. The diagonal direction follows theclosest pillar. The sheet lined with micro-pillar array has
distribution about local micro compliance. The behavior ofcell might depend on the local micro deformability of thescaffold.
The dislocation of the pillar is very small to detect deformationof the adhered cell. If myotube grows and deforms in thelarger scale, deformation of the scaffold might become larger.
Thickness of the sheet should be thinner than 0.02 mm to be
deformed with cells to detect behavior of cells.
The optical scattering by micro pillars on the scaffold disturbs
optical microscopic observation at cells on the scaffold. To
minimize the scattering, the micro-pillars should be shorter.
In the present study, the control of the volume of the medium is
not easy, because the volume of the medium is very small (0.2
cm3). With the mask, time for drawing is shortened. It iseasier to control the height of pillars with the mask.

5. CONCLUSION
The transparent micro-patterned scaffold has been designed toobserve the interaction between cultured cells in vitro. The
designed scaffold with micro-pillar and sheet has beensuccessfully fabricated by photolithography technique, andapplied to culture of L929 and C2C12. Cells are able to be
traced through the transparent scaffold. The designed scaffold

This work was supported by a Grant-in-Aid for Strategic
Research Foundation at Private Universities from the Japanese
Ministry of Education, Culture, Sports and Technology.

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