Myotube Cultured on Micro Coil Spring
Shigehiro HASHIMOTO, Hiroaki NAKAJIMA, Nobuaki AMINO, Kenta NODA
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/
ABSTRACT

2. METHODS

Myoblasts have been cultured on a micro coil spring to estimate
forces generated among the tissue of cultured myotubes. Amicro coil spring made of titanium wire of 0.085 mm was usedfor the scaffold for the cell culture. The coil has the dimension
as follows: 0.65 mm diameter, 0.05 mm pitch, 5 mm length.
C2C12 (mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse) was seeded at the concentration of
10000 cells per cm2. The cells around the coil were observedwith an inverted phase contrast microscope. The experimentshows the following results. Cells are able to adhere aroundthe coil, proliferate, differentiate into myotube, make cylindricallayer around the coil, and bridge between the pitches of coils.
The force, which is generated in myotubes pulling the wires
between pitches, is estimated to 0.01 N.

Micro CoilA micro coil spring (Hi-Lex Corp., Takarazuka, Japan) made oftitanium wire of 0.085 mm diameter was used for the scaffold
for the cell culture (Fig. 1). The coil has the dimension as
follows: 0.65 mm diameter, 0.15 mm pitch, 5 mm length.

Keywords: Biomedical Engineering, C2C12, Micro Coil andDifferentiation.

1. INTRODUCTION
Cell culture technique has been developed and several
methodologies have been clinically applied to regenerative
medicine [1]. The acceleration technique for orientation andproliferation of cells has been studied to make a biologicaltissue in vivo or in vitro [2-4]. Control methodology forbehavior of cells would be applied to regenerative tissue
technology: orientation, proliferation and differentiation.

Cell Culture
The micro coil spring was placed in the micro-plate of 24 wellsof flat bottom of polystyrene (Fig. 2). The internal diameter of
each well is 15 mm.
The sixteenth passage of C2C12 (Mouse myoblast cell line
originated with cross-striated muscle of C3H mouse) wasseeded at the concentration of 10000 cells per cm2.
C2C12 was cultured with the D-MEM (Dulbecco’s Modified
Eagle Medium) in the incubator for 37 days at 310 K with 5%of carbon dioxide gas. The medium contains FBS (fetalbovine serum) with volume percent of 10. The medium also
contains penicillin streptomycin with volume percent of one.
The whole body of the micro coil was dipped in the culture
medium during cell culture.

The effect of the surface of the scaffold on cell culture has beenstudied in the previous studies [5, 6]. Several factors, which
control adhesion of biological cells, have been studied in vitro[7].
When cells make a tissue, they might be pulling each other tokeep the morphology. The force generated in the tissue might
be estimated with the restoring force of the scaffold.
In the cell culture, myoblasts differentiate to myotubes, whichhave potential to generate repetitive contraction withstimulation of electric pulses.
When the scaffold hasresistance to contraction, the myotubes make movement ofrepetitive contraction with the scaffold.
In the present study, myoblasts have been cultured on a micro
coil spring to estimate forces generated among the tissue ofcultured myotubes.

Fig. 1: Micro coil spring.

slightly vibrated during handling of culture. Most of cells,
however, keep adhesion to the coil in three weeks of culture.
Several myotubes are observed around the coil in two weeks ofculture (Fig. 5). Most of myotubes tend to be oriented in theparallel direction to the wire of the coil.

Fig. 2: Micro-plate of 24 wells.

After four days of culture, the coil was moved to anothermicro-plate to be separated from the cells on the bottom of themicro-plate. The cells on the coil were successively cultured
for another four weeks. The medium was changed every twodays. The content of the medium was changed to D-MEM(Dulbecco’s Modified Eagle Medium) containing 5% HS (horseserum) and 1% penicillin streptomycin on the thirteenth day ofculture.

Fig. 3: C2C12 cultured 2 days around micro coil.
from left to right is 2 mm.

Dimension

The cells around the coil spring were observed with an inverted
phase-contrast microscope (IX71, Olympus Co., Ltd., Tokyo)
every day.
Spring Constant
The spring constant of the coil (k [N/m]) is calculated by Eq. 1.
k = F / x = G d 4 / (8 N D 3)

(1)

In Eq. 1, F is force [N], x is displacement [m], G is modulus oftransverse elasticity of the wire [Pa], d is diameter of wire [m]N is number of turns, and D is diameter of the coil [m].
E = 2 G (1+n)(2)

In Eq. 2, E is Young’s modulus, and n is Poisson’s ratio.
titanium, E = 1011 Pa and n = 0.3 make G =4 ×1010 Pa.

InFig. 4: C2C12 cultured 5 days on micro coil. Dimension fromleft to right is 2 mm.

In the micro coil spring of titanium used in the present study, G= 4 ×1010 Pa, d = 0.085 mm, N = 30 turns, and D = 0.65 mm
make k = 40 N/m.
When the dimension of a pitch of the coil varies 0.01 mm, thelocal force at one turn is 0.01 N.

3. RESULTS
Fig. 3 exemplifies C2C12 cultured for two days. In the figure,most of cells are on the bottom of the dish. After the coil was
moved to another micro plate, many cells are attached on thecoil.
Cells start to adhere to the coil in four days, and proliferate tocover the surface of the coil in five days of culture (Fig. 4). In
two weeks of culture, some of cells exfoliate, when the coil isFig. 5: C2C12 cultured 9 days on micro coil. Dimension fromleft to right is 2 mm.

(A)MyotubeMicro coil spring(B)
MyotubeFig. 6: C2C12 cultured 17 days on micro coil.
from left to right is 2 mm.

Dimension

Micro coil spring

Fig. 9: Hybrid actuator of micro coil spring with myotube: axial(A) and spiral (B) direction.

4. DISCUSSION

Fig. 7: C2C12 cultured 29 days on micro coil.
from left to right is 2 mm.

Dimension

After separation from the cells on the bottom of the micro-plate,most of cells are kept on the coil in the present experiment.
The cells proliferate, and differentiate keeping contact with thecoil.
When cells make tissue, a space for supplying medium isnecessary around cells. A coil has a spiral space along thewire. The space might give a path for the medium approach tothe cells.
In the previous studies, the several kinds of acceleration
technique to make orientation of cells were tried in vitro: withthe shear flow [2], with the gravitational force [3], with thenanofiber [8], or with the morphology of the surface [5, 6].
The micro coil spring gives a good scaffold for cell culture.
The spiral morphology of the coil might make the spiralorientation of myotubes (circle in Fig. 8).
C2C12 is able to adhere and proliferate on the surface of themicro coil spring of titanium. The cells are also able todifferentiate into myotubes around the coil spring.

Fig. 8: Myotube on micro coil.
1 mm.

Dimension from left to right isSome of the pitches of the coil decrease at the level of 0.01 mm,
when several myotubes are formed around the coil (Figs. 6 &7).

The coil spring deforms in proportion to the force. The force
generated in the muscle tissue cultured on the coil might beestimated by the displacement of the coil. The force generated
in the tissue in the present experiment is estimated to 0.01 N by
the displacement of 0.01 mm of the pitch of the coil. Theforce might be generated between the myotubes or in themyotube. The estimated force by the deformation of the coil isaxial direction of the coil. The direction of the force generated
in myotube might be longitudinal direction of the myotube,
which is oriented to the spiral direction of the coil (Fig. 8).

The movement of cultured myotubes is able to be controlledwith electric pulses supplied to the medium [9]. If the coilgenerates resistant force against the contractive force generated
by myotubes, the coil covered with myotubes has a potential to
make a repetitive contractile actuator [10-12]. The laser
system has been applied to measure the cyclic movement in thebiological system [13].
Titanium is one of the materials, which has been used forbiological application [14]. Titanium has been implanted tohuman body as a strut of valves, a root of teeth, pins inorthopedic treatment, and a part of joint.
The morphology of micro channel has simulated the lymph
system in the circulatory system in vivo. In several studies,permeability has been tried to control in designing artificial
vessels. The micro spring has a potential to be applied to thescaffold for the micro channel.
The experimental results will contribute to estimate forces
generated in the tissue of myotubes.

5. CONCLUSION
Myoblasts (C2C12) have been cultured on a micro coil spring
to measure forces generated among the tissue of cultured
myotubes. A micro coil spring (0.65 mm diameter, 0.05 mmpitch, 5 mm length) made of titanium wire of 0.085 mm wasused for the scaffold for the cell culture. The experimentshows that cells are able to adhere around the coil, proliferate,
differentiate into myotube, make cylindrical layer around thecoil, and bridge between the pitches of coils. The force, whichis generated in myotubes pulling the wires between pitches, canbe estimated with the spring constant.

6. ACKNOWLEDGMENTAuthors thank to Hi-Lex Corp. for supply of the micro coilspring. This work was supported by a Grant-in-Aid for
Strategic Research Foundation at Private Universities from theJapanese Ministry of Education, Culture, Sports andTechnology.

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