Simultaneous Measurement System for Local Medium pHand Movement of Contracting Myotubes in Vitro
Kousuke KIDA, Shigehiro HASHIMOTO, Eiji YAMADA, Masahide OKADA,Shuichi MOCHIZUKI, Toshia FUJISATO, Tomohiro SAHARA, Mieko OHSUGA
Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, 535-8585, Japanhashimoto@bme.oit.ac.jp http://www.oit.ac.jp/bme/~hashimotoandHajime OTANI
Cardiovascular Center, Kansai Medical University, Moriguchi, Japan
andKiyoshi YOSHINAKA
Department of Bioengineering, The University of Tokyo, Tokyo, Japan

ABSTRACTprobe is invasive and too large in scale to measure local pH
value in cell culture. To design a control method forenvironment to maintain a contractive movement of thebiological muscle tissue, the value of pH could be an indicator[3].

An optical measurement system has been designed to measure
Local pH of the medium and a movement of a contracting
cultured myotube simultaneously in vitro. A light source of alight-emitting diode and a helium neon laser was applied to theculture medium to measure pH in the medium and themovement of the contracting myotube, respectively. The
intensity of 558 nm wavelength of the transmitted light from thediode was measured to detect pH in the medium, which includes
phenol red. The intensity of 632.8 nm wavelength of thetransmitted light from the laser was measured to detect thecyclic contractile movement of the cultured myotube. C2C12(mouse myoblast) cells were cultured and differentiated tomyotubes. The cyclic contraction of the myotube was inducedwith electric pulses applied through the medium.
Themovement was measured with the laser beam under observation
with an inverted phase contrast microscope, while pH wasmeasured with the diode. The results show that the designedsystem was effective to measure local medium pH andmovement of contracting myotubes simultaneously in vitro.

Measurement System
The designed measurement system consists of three subsystems:a pH measurement system, a movement measurement system,
and a microscope system (Fig. 1). The whole system is locatedin a dark room.

Keywords: Biomedical Engineering, Muscle Cells, Cell Culture,
pH and Laser

The optical pH measurement system consists of a light source,concave lenses, convex lenses, a light path with aluminum

Most of medium, on the other hand, contains phenol red for pHindicator, which enables optical pH measurement between 6.8
and 8.4. In the present study, an optical measurement system
has been designed to measure pH of the medium adjacent tocontracting cultured myotubes in vitro. In the present study,
simultaneous measurement system has been designed for local
medium pH and for movement of contracting myotubes in vitro.

2. METHODS

1. INTRODUCTION
Biological muscle has a potential to realize a light small actuatorwith high efficiency. Recent progress in tissue engineering
realizes the muscle cell culture technique. The cultured muscle
cell has a potential to be applied to a micro actuator. The
previous studies show that mouse muscle cells differentiate tomyotubes and their periodical contraction is controlled withelectric pulses applied through the medium. The movement
can be measured with a laser beam [1].
The value of pH is one of the useful indicators to control thecontent of the medium for cell culture [2]. When the contentsof the medium vary with energy consumption during muscle
contraction, pH decreases to acidosis. The conventional pHFig. 1(a): Measurement system.

Detector
MicroscopeDetector

Detector
Laser
Culture DishLED
Fig. 1(b): Measurement system.

Fig. 4: Measurement of pH.

Fig. 2: Myotubes.
mm in the figure.

Distance between left and right is 0.8

cylinder, and a light-sensitive element. The light source ismade of nineteen green (558 nm wavelength [3]) light emitting
diode (LED) elements, which are connected each other inparallel electric circuit, and operated with an electric current of
300 mA (2.05 V) in total. The light-sensitive element is madeof a PIN photodiode, which converts light into an electriccurrent. The electric current is converted into an electric
potential with an amplifier. Data are collected with a datalogger. The light beam from LED does not have directivity.
The beam is concentrated with the lenses and cylindrical lightpath. The beam of 3 mm diameter is irradiated to the culture
dish. The intensity of the transmitted LED beam through theculture medium was detected with the photodiode.
The movement measurement system consists of a light source, aplate beam splitter, a total reflection mirror, a deflection plate,
two absorptive neutral density filters, two convex lenses andtwo photodiode detectors. A helium neon laser head with awavelength of 632.8 nm is used for the light source. The beamis split with the plate beam splitter to check the light source.
The intensity of the light decreases through the absorptive
neutral density filters. The beam is irradiated in the counter
direction of the beam of pH measurement system to avoid
interference between two light beams (Fig. 1). Fluctuating
intensity of the transmitted laser beam through the periodicallycontracting myotubes was detected with the photodiode.
The microscope system consists of a phase contrast inverted
microscope and a charge-coupled device (CCD) camera. The
movement of myotube was observed and recorded with themicroscope and with the camera at room temperature.

Fig. 3: A pair of electrodes on the cap of culture dish.

Myotubes
C2C12 (mouse myoblast cell line) cells were cultured with ahigh-glucose Dulbecco’s Modified Eagle’s Medium (D-MEM)
on a dish of 52 mm internal diameter to make myotubes in an4.0000

pH 8.43
pH 8.61
pH 8.25
pH 8.08
pH 7.05
pH 7.073.5000Absorbency

3.00002.5000

558nm

2.0000pH 8.65
pH 8.72
pH 8.11
pH 6.95
pH 7.87
pH 8.20

1.50001.00000.50000.0000-0.5000
190

290

390

490

590

690

790

890

990

1090

Wavelength (nm)Fig. 5: Relation between absorbency and wavelength for media of various pH.
Measurement of pH
Variation was made on medium pH in the culture dish withcarbon dioxide gas supply (Fig. 4). The culture dish with theculture medium (without cells) was put in a chamber connected
to a compressed gas cylinder of carbon dioxide. The value ofpH is confirmed with a conventional pH meter (Seven Multi,Metler Toledo, Switzerland), which has an electrode. Thetemperature around the dish was measured with a thermoelectriccouple.

Electric potential (mV)

236.0

235.5

235.0

To select the wavelength for the optical pH measurement system,
the transmitted light intensity between 190 nm and 1100 nm wasmeasured on the medium. Several samples of medium ofvarious pH were poured into the micro-cell, which was placedin a spectrophotometer (UVmini1240, Shimadzu, Kyoto, Japan).

234.5

234.0
6

78

9pHFig. 6: Relation between pH and output voltage of themeasurement system.

incubator. The D-MEM includes phenol red to indicate pH
between 6.8 and 8.4. The medium was replaced every twodays. In the first term, fetal bovine serum (FBS) was added tothe medium at a volume rate with 10 percent of FBS and 90
percent of D-MEM to accelerate proliferation.
In thesubsequent second term, FBS was switched to horse serum (HS)to induce differentiation (Fig. 2), before cells were proliferated
to a sub-confluent state on the culture dish. The second
medium consists of seven volume percent of HS and 93 volume
percent of D-MEM.
Electric Stimulation
Electric pulses were applied to the culture medium to control thecyclic contractile movement of myotubes. Two electrodes are
made of a platinum wire of 0.2 mm diameter (Fig. 3). To fix
the position of the tips of the electrode, the wire is inserted
through a curved glass pipe of 0.6 mm inside diameter. An
electronic stimulator is used to generate periodical rectangularpulses. Repetitive contractions of myotubes were induced withelectric pulses of one millisecond width. Variation was madein the period (between 0.2 s and 2 s) and the amplitude (between40 V and 60 V) of the cyclic electric pulses.

The output voltage of the pH measurement system wasmeasured, while the movement of myotubes were measured
with the laser system and observed with the microscope.

3. RESULTS
The muscle cells fused and differentiated to the myotubes, after
the FBS in the medium was replaced with HS (Fig. 2).
Fig. 5 shows the relation between absorbency and wavelength
for media of various pH. The peak of absorbency at thewavelength of 558 nm corresponds to the color displayed withthe phenol red.
The light from LED can be successfully concentrated into thespot of 3 mm diameter at a measurement position with thelenses. Through the designed light path with the lenses andwith the aluminum cylinder, enough intensity of light arrives atthe detector.
The temperature around the culture dish tends to increase withtime under lights: LED, laser and halogen lamp of themicroscope. When the temperature increases from 17 to 26
degrees centigrade, the output voltage of pH measurement
system decreases from 0.255 V to 0.235 V. The shifted value
with temperature is able to be compensated with the relation
between output voltage and temperature.
Fig. 6 shows the relation between pH and output voltage of themeasurement system after compensation about temperature.

Temperature [deg C]

Electric potential [mV]

Temperature [deg C]

Electric potential [mV]

295.5530.0

303.3529.0

295.5029.8

303.3028.8

295.4529.6

28.6

303.20295.4029.4

295.3529.2

295.30Potential
295.25303.25012

28.4

303.1528.2

303.10Temperature
34

29.0

303.050Potential
1Time [s]Fig. 7(a): Voltage tracings.

Period: 2 s.

Fig. 7(d): Voltage tracings.

Temperature [deg C]

Electric potential [mV]
303.5029.0

303.45303.40303.35

12
Time [s]Fig. 7(b): Voltage tracings.

3

Electric potential [mV]

Temperature [deg C]

28.8

28.6

303.6028.4

303.5028.2

303.45303.4004

28.6

303.55

Potential

Temperature

2Time [s]328.01303.0029.0302.9528.8

302.9028.6

302.8528.4

302.8028.2
Potential

Temperature

2Time [s]328.01

Period: 0.2 s.

Temperature [deg C]

Electric potential [mV]

302.7004

Period: 1 s.
Fig. 7(e): Voltage tracings.

302.75Period: 0.33 s.

303.65

28.0
028.0

28.8

28.2

303.20429.0303.30Potential Temperature3

303.7028.4

303.25

2Time [s]Temperature

Fig. 7(c): Voltage tracings.

4Period: 0.5 s.

The figure shows that electric potential varies linearly with pH
at the ratio of 0.33 mV/pH in the pH range between 6.8 and 8.4,
which is the color variation range for phenol red and isexperienced in the normal muscle cell culture medium.
The experimental results on electric stimulation to myotubes
show that the cyclic contractive movement of myotubes can besynchronously controlled with cyclic electric pulses conducted
through the culture medium. The cyclic movement wasconfirmed with the phase contrast inverted microscope.

Fluctuating intensity of the transmitted laser beam was traced
through the periodically contracting myotubes. The electric
signals of the photodiode were recorded in a computer with asampling frequency of one millisecond. In Fig. 7, the tracingsare displayed after filtration, which cut off the higher frequency
than 100 Hz. The spectrum was analyzed on the data in threeterms of the period of the corresponding electric stimulation
pulses.
Fig. 7 exemplifies output voltage tracings, when the period of
cyclic stimulation pulses is one second and the amplitude is 50
V. The peak spectrum was measured at 1 Hz, which coincides
with the stimulation period of one second (Fig. 8). The
spectrum of the higher frequency corresponds to the fast risingslope of the output voltage tracings. The signal corresponds tofaster contraction than sinusoid, where relaxation is slower inthe myotubes. Same kinds of data for contracting myotubes
were detected with the present experimental system, whenvariations were made in the period (between 0.2 s and 2 s) andthe amplitude (between 40 V and 60 V) of the cyclic electricpulses.
The change of pH was traced with electric potential of the pH
measurement system, while the contractile movement ofmyotubes was traced with the laser system. Figs. 7 and 8shows data of temperature, which are simultaneously measured
with the optical pH measurement system.

3025Power spectrum [dB]

Power spectrum [dB]30201510500246252015105080Frequency [Hz]2468Frequency [Hz]Fig. 8(a): Relation between power spectrum andfrequency of electric potential in Fig. 7(a).

Fig. 8(d): Relation between power spectrum andfrequency of electric potential in Fig. 7(d).

25302015105002468

Power spectrum [dB]

Power spectrum [dB]302520151050Frequency [Hz]02468Frequency [Hz]Fig. 8(b): Relation between power spectrum andfrequency of electric potential in Fig. 7(b).

Power spectrum [dB]

30Fig. 8(e): Relation between power spectrum andfrequency of electric potential in Fig. 7(e).

Relaxation

25Contraction

Potential

20mV

1510500246Time

8Frequency [Hz]tFig. 9: Myotube movement (upper) and electric potential.

Fig. 8(c): Relation between power spectrum andfrequency of electric potential in Fig. 7(c).
4. DISCUSSION

After injection of some amount of the carbon dioxide into thechamber, the cyclic contraction of myotubes in the chamber
stopped and the voltage signal of the transmitted light of LEDincreased. The increase of the voltage indicates the decrease of
pH in the designed measurement system. When the partialpressure of carbon dioxide decreased in the chamber in the
successive test, the cyclic contraction of myotubes started again
and the voltage signal of the transmitted light of LED decreased.
The decrease of the voltage indicates the increase of pH.

Two different wavelengths were applied to distinguish signal of
pH from that of movement at the simultaneous detection. The
two signals were successfully distinguished in the designed
system with two separated wavelengths: 558 and 632.8 nm.
The counter light path between pH and movement is effective todistinguish two transmitted signals.
Phenol red is contained in the conventional culture medium.
The color of the medium changes to yellow, when pH of themedium decreases. The color change enables the opticalmeasurement of pH in the medium. The designed opticalmeasurement system is useful to trace pH value of the culture
medium.
6. ACKNOWLEDGMENT

The intensity of the transmitted light depends on the depth ofmedium in the dish, which demands calibration to calculate
absolute value of pH in the optical sensor system.
An optical sensor is non-invasive. It has an advantage against
the conventional pH probe, because the conventional dipping
probe can cause contamination in the medium. The designed
optical measurement system also has a potential to realize
measurement for local pH at a micro scale.
The local contractive movement has been analyzed to explainthat of the tissue level [4]. The measurement system with laser
has a potential to be applied to local measurement for muscletissue. Cyclic contractive movement of myotubes might cause
cyclic fluctuation on the intensity of transmitted laser beam,
which enables measurement of movement (Fig. 9).
The optical pH measurement system can detect critical pH value
in the medium, when the cyclic contraction of myotubes in thechamber stopped during the test for variation of surrounding
partial pressure of carbon dioxide.
The system will contribute to control environment aroundmyotubes to maintain contractile movement [3, 5]. The
designed experimental system will contribute to develop amock-system for biological test instead of animal tests.

5. CONCLUSION

A system has been designed to measure movement ofcontracting myotubes and to measure local pH of medium
adjacent to the myotubes, simultaneously. The designed
system is effective to measure pH adjacent to contracting
cultured myotubes in vitro. The system will contribute tocontrol environment around myotubes to maintain contractile
movement.

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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