Design of Environment for Arrangementof Cultured Muscle Cells
Kenichi YAMASAKI, Shigehiro HASHIMOTO, Masahide OKADA, Kohei ONO,Toshia FUJISATO, Shuichi MOCHIZUKI, Masahiko YOSHIURA, Hiroshi TSUTSUI, Kenzo AKAZAWA
Department of Biomedical Engineering, Osaka Institute of Technology
Osaka, 535-8585, Japanhashimoto@bme.oit.ac.jp http://www.oit.ac.jp/bme/ABSTRACT
The effect of scaffold and flow on orientation of cultured muscle
cells has been studied to make muscle fiber in vitro. C2C12(Mouse myoblast cell line) cells were cultured with
High-glucose Dulbecco’s Modified Eagle’s Medium on a dish to
make muscle tubes. The scaffolds were designed with theoriented polypropylene fibers. In the other experiment, themedium was stirred to give flow stimulation on cells.
Differentiation was induced with horse serum. Orientation ofcells was observed with a phase-contrast microscope. The
experimental results show that the direction of cells can becontrolled with the direction of the scaffold fiber and the flow.

phosphate buffer solution including Laminin. In the latter case,the fibers were covered with type 1-A collagen dissolved inconcentrated culture medium. The fibers are located at 3 mmabove the bottom of the culture dish with the thick of silicone
rubber spacer. Cells were seeded on the scaffold and cultured.
When cells were seeded, a slide glass coated with cellulose was
located under the scaffold frame to avoid cell adhesion to thebottom of the dish (Fig. 2). The slide glass was removed in
two days of cultivation. C2C12 (Mouse myoblast cell line)
cells were cultured with Dulbecco’s Modified Eagle’s Medium
including ten percent of fetal bovine serum (FBS) and atibiotics.
The cells were seeded with density of 3000000 cells per dish.

Keywords: Biomedical Engineering, Muscle Cells, Scaffold,Flow, Differentiation and Orientation

1. INTRODUCTION
A biological muscle generates power even in a small unit,
collaborating with environment. The recent progress in cell
culture technique has realized muscle cell culture, which has
potential to be applied for an engineered actuator [1]. To makean efficient contractile unit with cultured muscle cells, cell
culture environment should be designed.
The effect ofmagnetic field on muscle cells [2] and the effect of surface
micro morphology on the alignment of skeletal muscle
myoblasts [3] have been studied in the previous studies. In thepresent study, the effect of scaffold and flow on orientation ofcultured muscle cells has been studied to make muscle fiber invitro.

Fig. 1: Scaffold frame with polypropylene fiber.

2. METHODS
Scaffold
Polypropylene fibers of 0.02 mm diameter were used as thescaffold for muscle cell culture. The fibers were soaked in an
ultrasonic washing machine filled with acetone to clean theirsurface, and dried. Every fiber was arranged parallel eachother with the chuck of silicone rubber of 3 mm thick. Afterthe distances between fibers were adjusted between 0.1 mm and
0.2 mm, the fibers were fixed on the stainless frame (Fig. 1).
Variations were made about coating on the fibers: Laminin (celladhesive glycoprotein constituting basement membrane) andcollagen. In the former case, the fibers were soaked inFig. 2: Arrangement of scaffold in culture dish.

Observed area
Silicone diskMediumFig. 3: Tilted rotating disk. Culture dish with (left) andwithout (right) silicone rubber sheet disk.

Flow

DishFig. 4: Tilted rotating disk. Culture dish with (left) andwithout (right) silicone rubber sheet disk.

The medium was replaced every two days. The cells were
cultivated for 16 days, and observed with a phase-contrast
microscope.
Reciprocating flow
A culture dish was placed on a plate, which generates seesaw
movement. A sheet of silicone rubber was sandwiched
between the dish and the disk for a nonslip strip. The tilted
angle was set to seven degrees, and the reciprocating speed wasset to thirty times per minutes in the test. A dish of 60 mm
diameter and a pentagon dish of 75 mm length were placed on
the disk. The reciprocating flow was generated in the medium
in the culture dish. The seesaw movement was started at thenext day from cells seeding, and kept for one week. C2C12(Mouse myoblast cell line) cells were cultured with Dulbecco’s
Modified Eagle’s Medium including ten volume percent of fetal
bovine serum (FBS) and atibiotics. The cells were seeded withdensity of 10000 cells per square centimeter. After four dayscultivation, ten volume percent of FBS was replaced with sevenvolume percent of horse serum to differentiate cells into
myotubes. The medium was replaced every two days. Thecells were observed with a phase-contrast microscope.
Rotational flow
A dish of 60 mm diameter was placed on the tilted rotating plate,which generates rotational seesaw wave movement (Fig. 3).
The tilted angle was set to six degrees, and the rotating speed
was set to fifty revolutions per minutes in the test. The seesaw
movement was applied for ten minutes at room temperaturewhen the medium was changed every two days.
Circumferential streamline was generated in the medium.
C2C12 (Mouse myoblast cell line) cells were cultured with
Dulbecco’s Modified Eagle’s Medium including ten volume
percent of fetal bovine serum (FBS) and atibiotics. The cells
were seeded with density of 3000 cells per square centimeter.
After four days cultivation, ten volume percent of FBS wasreplaced with seven volume percent of horse serum todifferentiate cells into myotubes. The medium was replaced
every two days. The cells were observed with a phase-contrast
microscope.
Narrowed rotational flow
A silicone rubber sheet disk of 3 mm thickness was placed in themiddle on the bottom of culture dish of 60 mm diameter (52 mm

Fig. 5: Cells cultivated for 2 days around Laminin coatedfiber. Bar indicates 0.1 mm.

Fig. 6: Cells cultivated for 2 days around collagen coatedfiber. Bar indicates 0.1 mm.
inner diameter) to restrict the space for the flow of the medium(Fig. 4). Variation was made in the disk’s diameter between 10Fig. 7: Cells cultivated for 16 days around Laminin coatedfiber. Bar indicates 0.1 mm.

Fig. 8: Cells cultivated for 16 days around collagen coatedfiber. Bar indicates 0.1 mm.
mm and 40 mm. The volume of the medium was adjusted not
to spread over the superior surface of the disk. One-way
rotational flow in the dish was generated with the platemovement of twenty revolutions per minute with six degrees
tilted angle. C2C12 cells were cultured with the same method
as the preceding section, while the plate was continuously
rotating at 37 degrees Celsius in an incubator. The cells wereseeded with density of 3000 cells per square centimeter. After
six days cultivation, ten volume percent of FBS was replacedwith seven volume percent of horse serum to differentiate cells
into myotubes. For comparison, cells were cultured in the dishwithout a silicone rubber sheet at the counter position on thedesk (Fig. 3).

3. RESULTS
Scaffold
Figs. 5-8 show the cells around the scaffolds.
Theexperimental results show that cells adhere to the scaffolds, andbridge fibers with sheet of cells in a week. Cells, although,show no orientation between scaffolds in Figs. 4-7. The
numbers of adhered cells were larger around fibers coated withFig. 9: Cells cultivated for 1 day with reciprocating flow.
Bar indicates 0.1 mm.

Fig. 10: Cells cultivated for 8 days with reciprocating flow.
Bar indicates 0.1 mm.
collagen than those coated with Laminin.
Flow
The experimental results with reciprocating flow show thatmuscle cells are proliferated, fused and differentiated tomyotubes, but their directions are random both in the middle
zone and in the peripheral zone of the dish regardless of theshape of the culture dish (Figs. 9 & 10). After flow stimulation
for ten minutes, cells were scattered at random, regardless ofculture days and of location in the dish (Figs. 11-14). The
result of one-way rotational flow shows regular array of
myotubes (Figs. 15-18), while that without silicone rubber sheet
shows random direction of myotubes (Fig. 19). The cells wereattached on the bottom of the dish in two days (Fig. 15) andarranged along the circumferential streamline around thesilicone rubber disk in six days (Fig. 16). The array of
myotubes grew around the silicone disk day by day, and thealignment curved to the radial direction of the disk as leaving
the disk (Figs. 17-18).

4. DISCUSSION
The present study shows following potentials. The collagenFig. 11: Cells in the middle zone cultivated for 2 days,before stimulation with flow for ten minutes. Bar
indicates 0.1 mm.

Fig. 13: Cells in the peripheral zone cultivated for 2days, before stimulation with flow for ten minutes. Bar
indicates 0.1 mm.

Fig. 12: Cells in the middle zone stimulated by flow forten minutes after cultivation for 2 days. Bar indicates
0.1 mm.

Fig. 14: Cells in the peripheral zone stimulated by flow
after cultivation for 2 days. Bar indicates 0.1 mm.

gel around fibers may supply environment where cells
adherence is accelerated. The silicone rubber spacer works to
distinguish the scaffold from the bottom of the culture dish. A
muscle cell colony can bridge between scaffold fibers, of which
distance is shorter than 100 micrometer. In the narrower space
between scaffold fibers, cells might line along the fiber.
Reciprocation of flow does not have significant effect to arrange
the direction of myotubes. The intermittent flow stimulation
for ten minutes does not have visible effect to uniform thearrangement of cells, either. An obstacle of silicone disk in themiddle part of culture dish is, on the other hand, is effective tonarrow the flowing space, where the rotational circumferential
flow occurs with swing motion of the tilted plate. That may
also help to restrict the secondary flow in the fluid. The flow
and the slope might affect arrangement of myotubes.

5. CONCLUSION
The effect of scaffold and flow on orientation of cultured musclecells has been studied to make micro actuator in vitro. The
experimental results show that cells tend to tilt to the directionof the scaffold fiber and the flow.

6. ACKNOWLEDGMENT
This work was supported by a Grant-in-Aid for Academic
Frontier from the Japanese Ministry of Education, Culture,
Sports and Technology.

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Yoshiura, K. Akazawa, M. Ohsuga, S. Uto, H. Otani and T.
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Fig. 15: Cells in the peripheral zone stimulated by flow after
cultivation for 2 days. 10 mm. Bar indicates 0.1 mm.

Fig. 18: Cells in the peripheral zone stimulated by flow after
cultivation for 11 days. 25 mm. Bar indicates 0.1 mm.

Fig. 16: Cells in the peripheral zone stimulated by flow after
cultivation for 6 days. 10 mm. Bar indicates 0.1 mm.

Fig. 19: Cells cultivated in flow for 5 days without
silicone rubber sheet. Bar indicates 0.1 mm.

Fig. 17: Cells in the peripheral zone stimulated by flow after
cultivation for 11 days. 15 mm. Bar indicates 0.1 mm.