Optical Measurement System for pH in Medium around
Contracting Myotubes in Vitro
Kosuke MATSUYOSHI, Shigehiro HASHIMOTO, Kosuke KIDA, Haruka IWATA
Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, 535-8585, Japanhashimoto@bme.oit.ac.jp http://www.oit.ac.jp/bme/~hashimoto

ABSTRACTAn optical measurement system has been designed to measure
local pH of the medium around contracting myotubes in vitro.
Variation was made for the light source to be applied to theculture medium: a light-emitting diode and a filtered mercury
lamp. The medium was placed on the stage of an inverted
phase contrast microscope, with which contracting myotubes
were observed. The intensity of the transmitted light throughthe medium from the light source was measured with aphotodiode to detect pH in the medium, which includes phenol
red. For calibration test, variation was made on pH in themedium with injection of carbon dioxide gas. The opticallydetected signal was referred to pH, which was measured with aconventional pH meter. The results show that the designed
system was effective to measure local medium pH aroundcontracting myotubes in vitro.
Keywords: Biomedical Engineering, Cell Culture, Optics andpH

1. INTRODUCTION
Biological muscle has a potential to realize a light small
actuator with high efficiency. Recent progress in tissue
engineering realizes the muscle cell culture technique. Thecultured muscle cell has a potential to be applied to a micro
actuator [1]. The previous studies show that mouse muscle
cells differentiate to myotubes and their periodical contraction
is controlled with electric pulses applied through the medium.
The movement can be measured with a laser beam [2].
The value of pH is one of the useful indicators to control thecontent of the medium for cell culture [3, 4]. When thecontents of the medium vary with energy consumption during
muscle contraction, pH decreases to acidosis.
The
conventional pH probe is invasive and too large in scale tomeasure local pH value in cell culture. To design a control
method for environment to maintain a contractive movement ofthe biological muscle tissue, the value of pH could be anindicator.
Most of medium, on the other hand, contains phenol red for pH
indicator, which enables optical pH measurement. In theprevious study, an optical measurement system in vitro has been
designed to measure repetitive contracting movement ofFig. 1: Green light emitting diode.

myotube and local pH between 6.8 and 8.4 around myotube
simultaneously [5].
In the present study, the opticalmeasurement system for pH in the medium has been modified
to improve signal to noise ratio.

2. METHODS
Optical property of medium
To select the wavelength for the optical pH measurement
system, the transmitted light intensity between 190 nm and1100 nm was measured on the medium. Several samples ofmedium with various pH were poured into the micro-cell, which
was placed in a spectrophotometer (UVmini1240, Shimadzu,Kyoto, Japan).
Measurement System
The optical pH measurement system consists of a light source, alight path, and a light-sensitive element. Variation was madefor the light source to be applied to the culture medium: alight-emitting diode and a filtered mercury lamp.
In the first system, the light source is made of a green(wavelength of 522 nm) light emitting diode (LED,LPF-05HGR-300N, Kilala-Factory, Japan), which is operated
with a direct electric current of 20 mA at 4.5 V (Fig. 1). The
optical cable (POC-15B) was applied to the light path to restrict
the light beam (Fig. 2). The light-sensitive element is made of

Fig.5: Optical characteristics of the super high-pressure
mercury lamp

Fig. 2: Optical cable.

Fig. 6: Optical characteristics of U-MNIB3 filter.

Fig. 3: Measurement system with LED.

a Si photodiode, which converts light into an electric current.
The intensity of the transmitted LED beam through the culture
medium was detected with the photodiode (Fig. 3). The
variation of electric characteristic of the photodiode wasdetected, and the relation between the variation and pH wasexamined. The beam of 3 mm diameter is irradiated to theculture dish.
In the second system, a super high-pressure mercury lamp wasused for the light source. The optical characteristics of thesuper high-pressure mercury lamp are illustrated in Fig. 4.
Before introduced to the medium, the light beam was filteredwith U-MNIB3 (Blue excitation, Fluorescence Mirror Unit,Olympus Corporation, Japan) or with U-DM-FI/TR2 (Greenexcitation, Fluorescence Mirror Unit, Olympus Corporation,Japan) (Fig. 4). Fig. 5 shows the optical characteristics of thesuper high-pressure mercury lamp. Fig. 6 shows the opticalcharacteristics of U-MNIB3 filter. Fig. 7 shows the opticalcharacteristics of U-DM-FI/TR2 filter. The light sensitiveelement is the same as that in the first system.

Fig. 4: Measurement system with filtered mercury lamp.

Fig. 9: Absorbency of medium in relation to wavelengthwith the variation of pH.
Fig. 7: Optical characteristics of U-DM-FI/TR2 filter.
Measurement of pH
Variation was made on pH (6.5-8.5) of medium in the culture
dish with carbon dioxide gas supply. The carbon dioxide gas
was produced by a chemical reaction with acetic acid andsodium bicarbonate liquid. The value of pH is checked with aconventional pH meter (Seven Multi, Mettler Toledo,Switzerland), which has an electrode (Fig. 8). The experiment
was performed at the room temperature of 25 degrees Celsius.

Fig. 10: Absorbency of medium in relation to pH at twowavelengths.
3. RESULTS
Fig. 9 shows the relation between absorbency of the mediumand wavelength with the variation of pH. The peak ofabsorbency at the wavelength of 558 nm corresponds to thecolor displayed with the phenol red. The result shows thatabsorbency also increases with pH at the wavelength of 522 nm(Fig. 10).
The light from LED can be successfully irradiated at ameasurement position with the optical cable. Fig. 11 shows
the relation between pH and electric resistance of the detector atthe LED test. The figure shows that electric property varies
linearly with pH between 6.3 and 7.8, which is the color
variation range for phenol red. The range includes pH value ofthe normal muscle cell culture medium.

Fig. 8: Measurement of pH.

Fig. 11: Detected signal in relation to pH at LED.

Detector
MicroscopeDetector

Detector
Laser
Culture DishLEDFig. 12: Detected signal in relation to pH at mercury lamp
filtered with U-MNIB3.

Fig. 14: Simultaneous optical measurement system.
The optical pH measurement system, for example, can detect
critical pH value in the medium, when the cyclic contraction of
myotubes in the chamber stopped during the test with variationof surrounding partial pressure of carbon dioxide [3]. The
optical system can be applied to the simultaneous measurement
system for pH and movement of cell (Fig. 14) [5].

Fig. 13: Detected signal in relation to pH at mercury lamp
filtered with U-DM-FI/TR2.
Fig. 12 and Fig. 13 show the relation between pH and electric
resistance of the detector at the filtered mercury lamp test. Fig.
12 shows the detected signal at the filtered with U-MNIB3.
Fig. 13 shows the detected signal at the filtered withU-DM-FI/TR2. Fig. 13 shows that that electric property varies
linearly with pH between 7.5 and 8.5.

4. DISCUSSION
The light source and path have been improved to increase thesignal to noise ratio.
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 opticalmeasurement system is useful to trace pH value of the culture
medium. Several perfusion systems have been developed fortissue culture [6-9]. The present measurement system has apotential to be applied to the bioreactor system.
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 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. Those
optical systems have also a potential to study muscle fibers orcells in detail [10, 11].

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.

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

REFERENCES.
[1] S. Hashimoto, S. Mochizuki, Y. Morita, H. Tsutsui, M.
Yoshiura, K. Akazawa, M. Ohsuga, S. Uto, H. Otani and T.
Fujisato, “Environmental Design for Muscle Cell Culture
with Magnetic Field”, Proc. 2007 Inaugural IEEE
International Conference on Digital Ecosystems andTechnologies (IEEE-DEST 2007), 2007, pp. 468-472.
[2] S. Hashimoto, S. Mochizuki, J. Takase and D. Inoue,“Measurement of periodical contraction of cultured muscle
tube with laser”, Journal of Systemics Cybernetics andInformatics, Vol. 7, No. 3, 2009, pp. 51-55.
[3] Q. Chen and D. R. Anderson, ”Effect of CO2 on Intracellular
pH and Contraction of Retinal Capillary Pericytes”,
Investigative Opthalmology & Visual Science, Vol. 38, No.

3, 1997, pp. 643-651.
[4] E. Yamada, S. Hashimoto, D. Inoue, H. Kondo, S.
Mochizuki, K. Yamasaki, T. Fujisato, M. Okada, H. Nakaoka
and M. Ohsuga (2008) “Medium control system for muscle
cell culture”, Proc. World Multi-Conference on Systemics
Cybernetics and Informatics, Vol. 2, 2008, pp. 120–123.
[5] K. Kida, S. Hashimoto, E. Yamada, M. Okada, S. Mochizuki,T. Fujisato, T. Sahara, M. Ohsuga, H. Otani and K.
Yoshinaka, “Simultaneous Measurement System for Local
Medium pH and Movement of Contracting Myotubes inVitro”, Proc. 13th World Multi-Conference on Systemics
Cybernetics and Informatics, Vol. 2, 2009, pp. 212-217.
[6] E. Cimetta, M. Flaibani, M. Mella, E. Serena, L. Boldrin, P.
DE. Coppi, and N. Elvassore, “Enhancement of Viability ofMuscle Precursor Cells on 3D Scaffold in a Perfusion
Bioreactor”, The International Journal of Artificial
Organs, Vol. 30, No. 5, 2007, pp. 415-428.
[7] G. H. Borschel, D. E. Dow, R. G. Dennis and D. L. Brown,“Tissue-Engineered Axially Vascularized Contractile
Skeletal Muscle”, Plastic and Reconstructive Surgery, Vol.

117, 2006, pp. 2235-2241.
[8] T. Sun, D. Norton, N. Vickers, S. L. McArthur, S. MacNeil,
A. J. Ryan and J. W. Haycock, “Development of a Bioreactor
for Evaluating Novel Nerve Conduits”, Biotechnology andBioengineering, Vol. 99, No. 5, 2007, pp. 1250-1260.
[9] T. Sun, D. Norton, J. W. Haycock, A. J. Ryan and S.
MacNeil, “Development of a Closed Bioreactor System forCulture of Tissue-Engineered Skin at an Air-LiquidInterface”, Tissue Engineering, Vol. 11, No. 11/12, 2005, pp.
1824-1831.
[10] Z. Ma, K. J. L. Burg, Y. Wei, X.-C. Yuan, X. Peng and B. Z.
Gao, “Laser-Guidance Based Detection of Cells withSingle-Gene Modification”, Applied Physics Letters, Vol.
92, 213902, 2008, pp. 1-3.
[11] F. H. Andrade, M. B. Reid, D. G. Allen and H. Westerblad,“Effect of Hydrogen Peroxide and Dithiothreitol onContractile Function of Single Skeletal Muscle Fibers from
the Mouse”, Journal of Physiology, Vol. 509.2, 1998, pp.
565-575.