Effect of Mechanical Stimulation on Orientation of Cultured Cell
Haruka HINO, Shigehiro HASHIMOTO, Masashi OCHIAI
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
shashimoto@cc.kogakuin.ac.jp http://www.mech.kogakuin.ac.jp/labs/bio/andHiromichi FUJIE
Human Mechatronic Systems, Faculty of System Design,
Tokyo Metropolitan University, Tokyo, Japan
ABSTRACTThe effect of mechanical stimulation on orientation of cultured
cells has been studied in vitro. Cells are exposed to two typesof mechanical stimulation: the gravity, and the flow. To apply
the excess gravitational force on the cultured cells, the cell
culture tube was set in a conventional centrifugal machine. To
apply the continuous shear flow on the cultured cells, the cell
culture dish with donut shaped canal was contained on theshaker. Three kinds of cells were alternatively used in the test:
L929 (fibroblast-like, mouse connective tissue), HUVEC(normal human umbilical vein endothelial cells), or C2C12(mouse myoblast cell line originated with cross-striated muscleof C3H mouse). The shape and the orientation of the cells
were observed with a phase contrast microscope during the cellculture. The experimental results show that HUBEC orients tothe stream line, although C2C12 tilts from the stream line andfrom the centrifugal direction.
Keywords: Biomedical Engineering, L929, HUVEC, C2C12,Cell Culture, Flow and Gravitational Force.

1. INTRODUCTION
Cell culture technique has been developed and several
methodologies have been clinically applied to regenerative
medicine. The acceleration technique for orientation andproliferation of cells has been studied to make tissue in vivo orin vitro [1-9]. Control methodology for orientation andproliferation of cells would be applied to the regenerative tissue
technology.
The mechanical stress is one of the interested points in theenvironment of cells, because they receive mechanical force invivo. The mechanical stress on cells might induce various
responses: deformation, migration, proliferation, anddifferentiation. Several methods have been designed to apply
the mechanical stress to cells [2-10].
A transmission point of the stress to a specimen is important.
In many studies, the stress is applied to a scaffold [2-5]. Whenfixation between the cell and the scaffold is not enough, thestress is not transmitted to the cell. A flow, on the other hand,
can be used to apply a stress field to a specimen [7-10]. Thespecimen directly receives the shear stress in the shear flow.
In the present study, the effect of mechanical stimulation onorientation of cultured cells has been studied in vitro.

2. METHODS
Cells are exposed to two types of mechanical stimulation: thegravity, and the flow.
Excess Gravitational ForceThe excess gravitational force was applied to cultured cells withthe centrifugal force (Fig. 1). Cells were cultured on thelateral wall of a tube, which was placed horizontally in theincubator (Fig. 2). Then, the tube (Fig. 3) was set on a rotor in
the centrifugal machine (MX105, Tomy Seiko, Co. Ltd, Tokyo,Japan) (Fig. 4). The rotor was rotated with the speed of 1100
revolutions per minute (rpm), which makes the excess
gravitational force of 100 G at the lateral wall of the culture
tube. To keep balance of the rotator in the centrifugal machine,
two tubes were placed in the counter position each other. On
the outer lateral wall of the tube, several lines are marked totrace the same area for the cell culture.

TubeMedium
Cell

Centrifugal force
Fig. 1: Centrifugal force (arrow) applied on cell culture in tube.

The constant excess gravitational force was applied on the cells
at 25 degrees Celsius. After morphological observation, thecells were cultured in the incubator again on the rest part of theday.

Fig. 2: Keeping horizontal position for cell culture on the innerlateral wall of tube.

Donut-Shaped Open Channel
A donut-shaped open channel system for the cell culture has
been designed to apply a vortex flow on cells in vitro. A
polystyrene culture dish was used.
A transparent
polydimethylsiloxane (PDMS) disk (30 mm diameter, 3 mmthick) is attached on the inner bottom of the culture dish torestrict the space for the flow of the medium (Fig. 5). The
PDMS disk is attached at the center of the culture dish of 52mm internal diameter with affinity between their surfaces
without adhesive.
The culture dish is placed on a plate, which inclines at 0.1 radof the horizontal plane (Fig. 6). The plate rotates to generate aswing motion (WAVE-SI, Taitec, Co., Ltd., Koshigaya). The
rotating speed of the plate is 30 revolutions per minute (rpm)(3.1 rad/sec). The motion produces a one-way counterclockwise vortex flow in the medium through the donut-shaped
open channel.
The continuously swinging plate is placed in an incubator (Fig.
7), where both the temperature of 310 K and the carbon dioxide
partial pressure of 5 percent are maintained.

Fig. 3: Cell culture in tube.

Fig. 5: PDMS disk attached on the bottom of culture dish.

Fig. 4: Rotor in centrifugal machine.

Fig. 6: Culture dish placed on shaker: counter clockwise flow.

3. RESULTS
The experimental results show that cells deform from the roundshape to the extended shape, which shows adhesion on the innerlateral wall of the polypropylene tube in 12 hours in theincubator. The appearance of the cell on the tube is similar tothat on the culture flask of polystyrene. The microscopic
observation shows, on the other hand, that six hours is notenough to adhere on the inner wall of the polypropylene tube.
Fig. 9 shows L929 cultured for 3 days with 100 G
centrifugation for 1 hour a day. The centrifugal force isapplied from right to left in Figs. 9-12. The experimental
result shows that the longitudinal axes of cells (L in Figs 9,10(B), 11(B), 12(B)) tend to tilt to the direction perpendicular tothe centrifugal force.
Fig. 7: Culture dish on shaker in incubator.
Figs. 10(A) and (B) show L929 before and after centrifugationof 100 G for 1 hour, respectively. Figs. 11(A) and (B) show
L929 before and after centrifugation of 100 G for 4 hours,respectively. Figs. 10(A) and 11(A) show L929 cultured for
12 hour on the lateral wall of the tube without centrifugation.
The figures show migration and deformation of L929.
Figs. 12(A) and (B) show C2C12 before and after centrifugationof 100 G for 3 min, respectively. Fig. 12(A) shows C2C12
cultured for 12 hour on the lateral wall of the tube withoutcentrifugation. The figures show migration and deformationof C2C12.

Fig. 8: Mark with interval of 0.5 mm to trace the same area ofculture.

After the cells were cultured for 12 hours without flow in theincubator, the cells were cultured under the flow for seven days.
Cell Culture
Three kinds of cells were alternatively used in the test: L929
(fibroblast-like, mouse connective tissue, RCB1422, Riken Bio
Resource Center, Tsukuba), HUVEC (normal human umbilicalvein endothelial cells), or C2C12 (mouse myoblast cell line
originated with cross-striated muscle of C3H mouse).

Fig. 13 shows HUVEC cultured in the donut-shaped open
channel for 12 hour without flow. Figs. 14(A) and (B) show
HUVEC cultured for 48 hour without and with flow,respectively. The result shows that proliferation decreases
with flow. Figs. 15(A) and (B) show HUVEC cultured for 6
days without and with flow, respectively. The experimental
result shows that HUBEC orients along the stream lines in 6days of culture (Fig. 15(B)).
Fig. 16 shows C2C12 cultured in flow for 5 days. The results
show that orientation (L in Fig. 16) of C2C12 tilts from thestream line (arrow). Fig. 17 shows C2C12 cultured in flow for
7 days. The results show that C2C12 differentiates tomyotubes under flow stimulation (the arrow in Fig. 17).

L929 was cultured with the E-MEM (Eagle’s minimal essentialmedium) containing 10% FBS (fetal bovine serum). HUVEC
was cultured with the EGM-2 (Endothelial Cell GrowthMedium). C2C12 was cultured with the D-MEM (Dulbecco’s
Modified Eagle Medium) containing 10% FBS. The medium
containing penicillin and streptomycin was refreshed every twodays.
Before the centrifugal test, the cells were cultured on the innerlateral wall of the polypropylene tube, keeping the horizontal
position in the incubator for 12 hours.
Morphological Study
Morphology of cells was observed with an inverted
phase-contrast microscope (IX71, Olympus, Tokyo). To trace
the same area in the serial cultivation, crossing lines have beenmarked on the outside of the wall of culture tubes and dishes(Fig. 8).

L

Fig. 9: L929 cultured for 3 days with 100 G centrifugation for 1
hour a day. Centrifugal force: from right to left (arrow).
Dimension from left to right is 1 mm.

L

Fig. 10(A): L929 before centrifugation. Dimension from left
to right is 0.5 mm.

Fig. 11(B): L929 after centrifugation: 100 G, 4 hour.
Dimension from left to right is 0.5 mm.

L

Fig. 12(A): C2C12 before centrifugation.
Fig. 10(B): L929 after centrifugation: 100 G, 1 hour.
Dimension from left to right is 0.5 mm.

L

Fig. 12(B): C2C12, after centrifugation: 100 G, 3 min.
Dimension from left to right is 1 mm.
Fig. 11(A): L929 before centrifugation. Dimension from left
to right is 0.5 mm.

Fig. 13: HUVEC cultured for 12 hour. The bar shows 0.1mm.

Fig. 14(A): HUVEC cultured for 48 hour without flow. Thebar shows 0.1 mm.

Fig. 15(A): HUVEC cultured for 6 day without flow.
shows 0.1 mm.

The barFig. 15(B): HUVEC cultured in flow for 6 day: arrow shows
direction of flow. The bar shows 0.1 mm.

LFig. 14(B): HUVEC cultured in flow for 48 hour: medium
flows from top to bottom. The bar shows 0.1 mm.

Fig. 16: C2C12 cultured in flow for 5 days: medium flows fromtop to bottom. Dimension from left to right is 1 mm.

5. CONCLUSION
The effect of gravitational force and flow on orientation of cellhas been studied in vitro. To apply excess gravitational force
on cells, the cell culture tube was set in a conventionalcentrifugal machine. To apply continuous shear flow on cells,
the cell culture dish with the donut shaped canal was containedon the shaker. The experimental results show that HUBEC
orients to the stream line, although C2C12 tilts from the streamline and from the centrifugal force line.

6. ACKNOWLEDGMENT
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.
Fig. 17: C2C12 cultured for 7 days in flow: medium flows from
top to bottom. Dimension from left to right is 0.5 mm.
REFERENCES
4. DISCUSSION
It is not easy to estimate the shear stress value on the wall in thepresent experiment, because the medium has the free surface.
The parallel piped chamber is convenient to observe theresponse of cells under controlled shear stress [8].
When mechanical stimulation is applied to the scaffold, thewhole stimulation cannot always be transmitted to the cells.
To apply mechanical stimulation to the cells, centrifugal force
or shear flow is used alternatively in the present study.
Both acceleration of proliferation and orientation of cells areimportant targets in the research field of regenerative medicineon the cultured biological tissue. The previous study showsthat electric stimulation enhances differentiation of muscle cells[1].
Another study shows that mechanical stimulation
improves a tissue-engineered human skeletal muscle [2].
The previous studies show that a mechanical field, on the otherhand, governs behavior of cells. The shear flow governs theorientation of endothelial cells [7, 9]. The shear stress affects
the orientation of the smooth muscle cells in the biologicaltissue [3]. The direction of the mechanical field affects
fibroblasts [5].
Too strong mechanical stimulation damages cells.
The
moderate mechanical stimulation, on the other hand, might
accelerate differentiation of cells [6].
The mechanicalstimulation can decrease proliferation of cells [6]. The
mechanical stress also exfoliates several cells, which makes
vacancy around the adhesive cell. The differentiation might be
optimization of cells to the changing environment. The
mechanical stress can accelerate differentiation of C2C12 to
make myotubes (Fig. 17).
The effect of shear flow on orientation of cells depends on thekinds of cells [7]. Although HUBEC orients along the streamlines, C2C12 tilts from the stream lines to make myotubes.
The previous study shows orientation of cells perpendicular tothe stretch direction [4].
The results of the study will contribute to acceleration techniquein regenerative medicine[1] S. Hashimoto, F. Sato, R. Uemura and A. Nakajima, “Effectof Pulsatile Electric Field on Cultured Muscle Cells in Vitro”,
Journal of Systemics Cybernetics and Informatics, Vol.
10, No. 1, 2012, pp. 1-6.
[2] Y. Akiyama, R. Terada, M. Hashimoto, T. Hoshino, Y.
Furukawa and K. Morishima, “Rod-shaped Tissue
Engineered Skeletal Muscle with Artificial Anchors toUtilize as a Bio-Actuator”, Journal of Biomechanical
Science and Engineering, Vol. 5, No. 3, 2010, pp. 236-244.
[3] K. Nakagawa, N. Morishima and T. Matsumoto, “Effect ofThree-Dimensional Culture and Cyclic Stretch Stimulation
on Expression of Contractile Protein in Freshly Isolated Rat
Aortic Smooth Muscle Cells”, Journal of Biomechanical
Science and Engineering, Vol. 4, No. 2, 2009, pp. 286-297.
[4] L. Terracio, B. Miller and T. Borg, “Effects of CyclicMechanical Stimulation of the Cellular Components of theHeart: in Vitro”, In Vitro Cellular & DevelopmentalBiology, Vol. 24, No. 1, 1988, pp. 53-58.
[5] J.H.-C. Wang, G. Yang, Z. Li and W. Shen, ”Fibroblast
Responses to Cyclic Mechanical Stretching Depend on Cell
Orientation to the Stretching Direction”, Journal ofBiomechanics, Vol. 37, 2004, pp. 573-576.
[6] S. Motoda, S. Hashimoto, T. Iwagawa and A. Nakajima,“Effect of Excess Gravitational Force on Cultured Myotubesin Vitro”, Proc. 15th World Multi-Conference onSystemics Cybernetics and Informatics, Vol. 2, 2011, pp.
118-123.
[7] S. Hashimoto and M. Okada, “Orientation of Cells Culturedin Vortex Flow with Swinging Plate in Vitro”, Journal ofSystemics Cybernetics and Informatics, Vol. 9, No. 3,
2011, pp. 1-7.
[8] S. Hashimoto, F. Sato, K. Oya, H. Fujie, H. Iwata and Y.
Sakatani, “Responses of Cells to Flow in Vitro”, Proc. 16th
World Multi-conference on Systemics Cybernetics andInformatics, Vol. 2, 2012, pp. 91-96.
[9] Y. Sugaya, N. Sakamoto, T. Ohashi and M. Sato,“Elongation and Random Orientation of Bovine Endothelial
Cells in Response to Hydrostatic Pressure: Comparison withResponse to Shear Stress”, JSME International Journal,Series C, Vol. 46, No. 4, 2003, pp. 1248-1255.
[10] P. Uttayarat, M. Chen, M. Li, F.D. Allen, R.J. Composto
and P.I. Lelkes, “Microtopography and Flow Modulate theDirection of Endothelial Cell Migration”, Am. J. Physiol.
Heart Circ. Physiol., Vol. 294, 2008, pp. H1027-H1035.