Movement of Cultured Myotube with Electrical Stimulation

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1 Movement of Cultured Myotube with Electrical Stimulation Hideo KONDO, Shigehiro HASHIMOTO, Kenichi YAMASAKI, Kohei ONO, Masahide OKADA, Toshia FUJISATO, Hiroyuki KOBAYASHI, Shuichi MOCHIZUKI, Mieko OHSUGA, Masahiko YOSHIURA, Hiroshi TSUTSUI, Kenzo AKAZAWA, Toshikazu KAWAI, Sadahito UTO, Katsuyoshi TSUJITA, Eiji YAMADA, Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, , Japan and Tetsuji YAMAOKA Department of Biomedical Engineering, Research Institute of National Cardiovascular Center, Suita, Japan ABSTRACT Repetitive contraction of cultured muscle tubes has been controlled in vitro with periodical electrical stimulation. C2C12 (Mouse myoblast cell line) was cultured with High-glucose Dulbecco s Modified Eagle s Medium on a dish to make muscle tubes. Differentiation was induced with horse serum. Repetitive contraction of the tube was generated by electric pulses through electrodes of platinum, and observed with a phase-contrast microscope. The cells were proliferated and differentiated to micro-tubes in six days. The periodical contraction of the tube was observed with the periodical electric pulse between.5 Hz and Hz. A Tetanic contraction was occurred with a frequency higher than 5 Hz. Contraction was controlled with lower amplitude and with shorter pulse width of the electric pulse, when the direction of the electric field is parallel to the longitudinal axes of the tube. Keywords: Biomedical Engineering, Cell Culture, Myotube, Electrical Stimulation, Electric Field Direction and Frequency 1. INTRODUCTION A biological muscle is a light compliant actuator at high efficiency compared with an engineered mechanical actuator. Cell culture technique, on the other hand, has been developed, and myoblast has been cultured in previous studies [1]. The cultured muscle cell has a potential to develop a new actuator [2]. designed, and repetitive contractile movement of myotubes has been measured with variation of frequency, width and peak value of pulses. 2. METHODS Myotube C2C12 (Mouse myoblast cell line) was seeded on the dish of 6 mm diameter coated with collagen, so that the density is adjusted to 1 cells per square millimeter. The cells were cultured in an incubator with High-glucose Dulbecco s Modified Eagle s Medium (HG-DMEM) including fetal bovine serum (FBS) in the ratio of 1 volume percent. When the cells increased to confluent state, FBS was changed to seven volume percent of horse serum (HS) to induce fusion of muscle cells. The medium was replaced every two days, when the cells were observed with a phase-contrast microscope. Electrode Electrodes were made of platinum plate lined with a polycarbonate plate (Fig. 1). The width and the depth of the electrode are 17 mm and 4 mm, respectively. Four plates of electrode are located on the 24 mm length fringe of a square frame made of polycarbonate (Fig. 2). To evaluate the effect Repetitive contraction of cultured muscle tubes has been controlled in vitro with periodical electrical stimulation [, 4]. To evaluate the performance of the cultured muscle tubes as an actuator, both an experimental system for stable electric stimulation and measurement methodology for the contractile movement of muscle tubes are necessary. In the present study, the electric pulse stimulation system in vitro with a stable electric field for cultured myotubes has been Fig. 1: Electrode.

2 diameter (Fig. ). Electric Potential Distribution Distribution of electric potential in the medium contained in the culture dish was measured. Fig. 4 shows the needle electrode of the platinum wire to explore electric potential in the medium. The electrode was fixed at the jig, which is placed on the stage to adjust its two dimensional position. Fig. 2: Four plates of electrode located on a square frame. 6 mm Dish Frame of electrode Five milliliter of high-glucose Dulbecco s modified Eagle s medium (HG-DMEM) including fetal bovine serum (FBS) was filled into the culture dish of 6 mm diameter. The electrode plates were placed in the medium, and the periodical electric pulses were applied. The pulses are rectangular with the following parameters: a period of one second, a width of 2 millisecond, a peak potential of 1 V, and a base potential of V. The needle electrode was dipped into the medium by 2 mm depth from the surface and the electric potential was measured every 2 mm from the anode plate to the cathode plate (Fig. ). The distribution of the electric potential in the medium was measured by shifting the measurement line of the needle electrode every 2 mm along the parallel direction to the electrode plate. The electric potential distribution was also measured in the medium, which includes C2C12 cells. After eight days cultivation, medium with C2C12 cells was used for measurement of electric potential distribution. mm Needle electrode 2 mm 6 mm Dish Fig. : Culture dish and frame of electrode. mm Electrical Stimulation On the eighth day of culture, the electrodes were placed in the dish. To stabilize hydrogen-ion concentration during the measurement, the medium was replaced to HG-DMEM including ten volume percent of FBS, antibiotic, and HEPES (2-[4-(2-Hydroxyethyl)-1-piperadinyl] ethane sulfonic acid). The directions of myotubes were random on the culture dish. After a myotube, of which longitudinal axis was parallel to the electric field, was selected and stimulated with electric pulses, the pair of the electrodes was switched to the other pair to change the direction of the electric field, which is perpendicular to the longitudinal axis of the myotube. The tests were performed at 7 degrees Celsius. The electric stimulation test system consists of a waveform generator, an amplifier, electrodes, an oscilloscope, a phase-contrast microscope and an incubator. In the first test, variation was made in pulse frequency. The electric pulses applied to the medium had following parameter: a base value of zero volt, a peak value of twenty volts, a duration of ten millisecond, a period between.1 and 2 seconds. Fig. 4: Needle electrode. of direction of electric field on the movement of myotube, two pairs of parallel plate electrode were prepared, of which the position are perpendicular each other. The frame was dipped in the middle part of medium in a culture dish of 6 mm In the second test, the minimum value to generate repetitive contractive movement of the myotubes was traced on both the width and the peak electric current of pulse at a constant period of one second, while the movement was observed with the phase-contrast microscope. The electric current was calculated with a voltage between both ends of a resistance of hundred ohms, which is on the serial connection to the electrodes. Analysis of Movement During the stimulation, movement of myotubes was observed with a phase-contrast microscope, and recorded with a computer.

3 The captured images were converted to grayscale picture. The mean value of the grayscale grade at the target area was traced, and spectrum of fluctuation with time was analyzed.. RESULTS Fig. 5 shows the electric potential distribution in the medium between two electrodes in the culture dish. The electrodes are located both on the left rim and on the right rim in Fig. 5. The position is marked as the distance from the center in millimeter, and the electric potential is illustrated with each colored band for every.5 V in Fig. 5. The experimental results show that the electric potential is approximately symmetry. Fig. 6 shows electric potential along the line from left to right in Fig. 5, which is rectangular to the electrode plate. The figure shows that the electric potential decreases linearly with the distance from the anode electrode plate. Fig. 7 shows the electric field along symmetric axis, which runs along the centerline of the dish from one electrode to the other. The experimental results show that the electric field is highest at the middle, and that the variation of the electric field is less than ten percent within the area of 5 mm from the center of the square of the frame Fig. 5: Electric potential distribution in medium: numbers indicate distance in mm, and potential range in volts. (V) Fig. 6: Electric potential in relation to distance from the center at each line perpendicular to the anode plate. Electric field (V/mm) Fig. 7: Electric field in relation to distance from the center at middle line perpendicular to the anode plate Fig. 8: Electric potential distribution in medium with cells: numbers indicate distance in mm, and potential range in volts. (V) Fig. 9: Electric potential in medium with cells in relation to distance from the center at each line perpendicular to the anode plate. Figs. 8-1 show the experimental results on the medium with C2C12 cells cultured for 8days: electric potential distribution, the electric potential and the electric field, respectively. The results are approximately same as that without cells.

4 Electric field (V/mm) Fig. 1: Electric field in medium with cells in relation to distance from the center at middle line perpendicular to the anode plate. Fig. 1: C2C12 cells cultured for 6 days: scale-bar shows.1 mm. Fig. 11: C2C12 cells cultured for 2 days: scale-bar shows.1 mm. Fig. 14: C2C12 cells cultured for 8 days: scale-bar shows.1 mm Fig. 12: C2C12 cells cultured for 4 days: scale-bar shows.1 mm. Figs show cultured cells observed with the phase-contrast microscope, cultured for 2, 4, 6, 8, 1, 12 days, respectively. Fig. 11 shows that cells proliferate to the confluent state. Figs show that cells differentiate and fuse to make myotubes. Cells peeled, and the number of cells decreased after 1 days (Figs. 16). Fig. 15: C2C12 cells cultured for 1 days: scale-bar shows.1 mm. The experimental results with constant peak value and width of pulse were illustrated in Figs The mean value of grayscale grade fluctuated with electric pulse stimulation (Fig. 17). The peak frequency spectrum was synchronized with that

5 Fig. 16: C2C12 cells cultured for 12 days: scale-bar shows.1 mm. Mean value of grayscale Fig. 19: Spectrum of fluctuation on grayscale tracings during electric stimulation of 1 Hz, when direction of Longitudial 2 Longitudinal Time (s) Fig. 17: Mean value of grayscale tracings during electric stimulation, when direction of electric field is parallel (longitudinal) and perpendicular (transverse) to the axes of myotubes Frequency (Hz) Fig. 2: Spectrum of fluctuation on grayscale tracings during electric stimulation of Hz, when direction of Longitudinal Longitudinal Frequency (Hz) Frequency (Hz) Fig. 18: Spectrum of fluctuation on grayscale tracings during electric stimulation of.5 Hz, when direction of of electric pulse between.5 and 5 Hz (Figs ), while the spectrum was not calculated at 1 Hz because of the limitation of the sampling frequency of the image in the measurement system. The periodical contraction of the myotube was observed with the periodical electric pulse between.2 and 2 seconds, when the electric field was parallel to the longitudinal axis of the myotube. The contraction was not observed, however, when the electric field was perpendicular to the Fig. 21: Spectrum of fluctuation on grayscale tracings during electric stimulation of 5 Hz, when direction of longitudinal axis of the myotube. Fig. 22 shows relationships between the minimum electric current and the minimum pulse width to generate movement of myotubes. When the direction of the electric field is parallel to the longitudinal axes of the myotube, repetitive contractive movement of myotube was controlled with smaller amplitude and with shorter width of the electric pulse.

6 Current (ma) Longitudinal Approximate Longitudinal Approximate longitudinal axes are perpendicular to the electric field. The present study shows that repetitive contractile movement of cultured muscle tube is controlled with smaller amplitude and with shorter width of the electric pulse, when the direction of the electric field is parallel to the longitudinal axes of the muscle tube. 5. CONCLUSION A contractile muscle tube has successfully been cultured and the repetitive contraction of the tube with electric stimulation has been observed in vitro. The results show that movement of tubes is larger along the electric field than that of those in the perpendicular to the electric field Pulse width (ms) Fig. 22: Relationship between the minimum electric current and the minimum pulse width to generate movement of myotubes. 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 4. DISCUSSION A stable electric field is necessary to measure contractile movement of cultured myotube with electric stimulation. The designed experimental system with platinum plates realizes a uniform electric field in the middle part of the medium. The cells were proliferated and differentiated to micro-tubes in six days. The repetitive contractile movement was traced with the grayscale grade fluctuation, while the cultured muscle tubes were stimulated with electric pulses. The experimental results show that the frequency of contraction is synchronized with that of electric pulse between.5 Hz and Hz. A tetanic contraction was occurred with a frequency higher than 5 Hz. In a physiological test, the minimum value to generate contraction of muscle was traced both on the width and on the peak electric current of pulse []. In the present study, the minimum values have been traced on muscle tubes, of which longitudinal axes are parallel to the electric field. The values have been compared to those on muscle tubes, of which [1] S. G. Carroll, R. J. Triolo, H. J. Chizeck, R. Kobetic and E. B. Marsolais, Tetanic Responses of Electrically Stimulated Paralyzed Muscle at Varying Interpulse Intervals, IEEE Transactions on Biomedical Engineering, Vol. 6, No. 7, 1989, pp [2] S. Hashimoto, H. Tsutsui, S. Mochizuki, M. Yoshiura, S. Uto, K. Akazawa, M. Ohsuga, H. Kobayashi, T. Kawai, K. Yamasaki, H. Kondo, K. Imoto, J. Takase, M. Okada, H. Otani, T. Fujisato and K. Yoshinaka, Environmental Design of Muscle Cell Culture for Micro-actuator, Proc. 11th World Multi-conference on Systemics Cybernetics and Informatics, Vol. 4, 27, pp. -5. [] R. G. Dennis, P. E. Kosnik II, M. E. Gilbert and J. A. Faulkner, Excitability and Contractility of Skeletal Muscle Engineered from Primary Cultures and Cell Lines, Am J Physiol Cell Physiol, Vol. 28(2), 21, pp. C288-C295. [4] M. Marotta, R. Bragos and A. M. Gomez-Foix, Design and Performance of an Electrical Stimulator for Long-Term Contraction of Cultured Muscle Cells, BioTechniques, Vol. 6(1), 24, pp