Measurement of Periodical Contraction of Cultured Muscle Tube with Laser

Transcription

1 Measurement of Periodical Contraction of Cultured Muscle Tube with Laser Masahide OKAA, Shigehiro HASHIMOTO, Jun TAKASE, Mieko OHSUGA, Kana NAKAMURA, Kenzo AKAZAWA, Shuichi MOCHIZUKI, Hiroyuki KOAYASHI, Toshia FUJISATO, Toshikazu KAWAI, Sadahito UTO, Katsuyoshi TSUJITA, Eiji YAMAA, Hideo KONO epartment of iomedical Engineering, Osaka Institute of Technology, Osaka, , Japan Hajime OTANI Cardiovascular Center, Kansai Medical University, Moriguchi, Japan Kiyoshi YOSHINAKA epartment of ioengineering, The University of Tokyo, Tokyo, Japan Tetsuji YAMAOKA epartment of iomedical Engineering, National Cardiovascular Center, Suita, Japan ASTRACT Periodical Contraction of cultured muscle tube has been measured with laser in vitro. C2C12 (Mouse myoblast cell line) was cultured with High-glucose ulbecco s Modified Eagle s Medium on a dish to make muscle tubes. ifferentiation was induced with horse serum. Repetitive contraction of the tube was generated by electric pulses lower than sixty volts amplitude with one milli-second width through electrodes of platinum, observed with a phase-contrast microscope. A laser beam of nm wavelength was restricted to mm diameter, irradiated on the bottom of the culture dish. Fluctuating intensity of the transmitted laser beam through the contracting muscle tubes was measured, its spectrum was analyzed. The results show that the electric pulses between 0.2 s 2 s. Keywords: iomedical Engineering, Muscle Cell, Measurement, Laser, Periodical Contraction, Synchronization Spectrum 1. INTROUCTION A biological muscle has potential to realize a light actuator with high efficiency. Cell culture technique, on the other h, has enabled muscle cells culture in vitro recently [1, 2]. Muscle cells differentiate to muscle tubes reveal their contractile function in vitro [3, 4]. The property of the muscle tubes has been examined in vitro in the previous study [5]. In the present study, an experimental system with a laser beam has been designed to measure micro repetitive movements of cultured muscle tubes in vitro. 2. METHOS Muscle tube C2C12 (Mouse myoblast cell line, Fig. 1) was cultured with High-glucose ulbecco s Modified Eagle s Medium (-MEM) on a dish of 60 mm diameter to make muscle tubes. The bottom of the dish is coated with collagen type I. Cells were seeded with two thous cells per one square centimeter, cultured in an incubator. The medium was replaced every two days. In the first term, fetal bovine serum (FS) was added to the medium with the volume rate in 10 percent of FS 90 percent of -MEM to accelerate proliferation. In the second term, FS was switched to horse serum (HS) to induce differentiation, before cells were proliferated to a sub-confluent state. The medium consists of seven percent of HS 93 percent of -MEM. The cells were observed with a phase-contrast microscope.

2 two convex lenses two detectors. A helium neon laser head with a wavelength of nm (Sigma Koki, LHP) is used for the light source. The beam is split with a plate beam-splitter, the alignment of the beam is adjusted with the CC camera. The diameter of the laser beam decreases to mm through the convex lens, when the beam incidents the culture plate. A thin black film with a hole of 0.45 mm diameter is attached on the bottom of the culture dish to mark the observation area (Fig. 3). Intensity of the transmitted laser beam through the culture dish is measured with the photodiode detector. The both terminals of the diode are connected to a resistance of one mega-ohm, voltage between two terminals is measured. The digitized voltage value is traced with a computer through an analog-to-digital converter with a sampling interval of 1 millisecond (Fig. 4). The spectrum of the traced data of 4096 (for four seconds approximately) was analyzed. Fig. 1: Myoblast, C2C12. φ0.45mm Cell L Fig. 3(a): Film attached on the bottom of the culture dish. M C F F L C P S M Fig. 2(a): Laser system: S, light source;, beam splitter; C, CC camera; M, total reflection mirror; P, deflection plate; F, absorptive neutral density filter; L, convex lens;, detector.. Fig. 3(b): Microscope view through a film attached on the bottom of the culture dish. Fig. 2(b): Laser system. Measurement system A measurement system (Fig. 2) was manufactured with laser. The system consists of a light source, three beam splitters, two charge-coupled device (CC) cameras, two total reflection mirrors, a deflection plates, two absorptive neutral density filters, Fig. 4: Electric pulse generator (bottom), data storage computer, phase-contrast microscope (right).

3 Fig. 5: Electrodes. Fig. 8: Multiple layers of muscle tubes. The electronic stimulator (Nihon Kohden, SEM-4201) was used to generate periodical rectangular pulses (Fig. 4). Repetitive contraction of muscle tubes was induced with electric pulses of one milli-second width. Variation was made in the period (between 0.1 s 2 s) the amplitude (between 40 V 60 V) of the repetitive pulses. 3. RESULTS The sub-confluent state was observed in five days of cultivation. The muscle cells fused differentiated to the muscle tubes, after the FS in the medium was replaced with HS (Fig. 7). Fig. 6: Culture dish with electrodes. Fig. 7: Muscle tubes, C2C12. Electric stimulation Electrode is made of platinum wire of 0.2 mm diameter. To fix the position of the tip of the electrode, the wire is inserted through a curved glass pipe of 0.6 mm inside diameter of 1.0 mm outside diameter (Fig. 5). A block of polymethyl methacrylate is used to fix each electrode at the fringe of the culture dish. To generate an electric field in the culture medium, two electrodes were dipped in the medium with the distance of 30 mm each other in a counter position (Fig. 6). After eleven days of cultivation, several contractive tubes were observed, when the electric pulses were applied. The contractive muscle tube was formed on the second layer from the bottom of the culture dish (Fig. 8). The contractive muscle tubes were observed between the eleventh day the thirty-eighth day. of the muscle tube was observed with the phase-contrast microscope, when the periodical electric pulses between 0.5 s 2 s were applied. Fluctuating intensity of the transmitted laser beam was measured through the contracting muscle tubes, its spectrum was analyzed. The digitized intensity tracings their spectrum are exemplified at period of 2 s in Fig. 9 Fig. 10, respectively. The peak spectrum was measured at the same frequency as that of electric pulses. The results show that the electric pulses between 0.2 s 2 s. The peak spectrum was not observed at the pulse interval of 0.1 s. The results show that the fluctuating amplitude of the intensity decreases with frequency of the stimulating pulses cannot be detected at the frequency of 10 Hz. The fluctuating amplitudes did not significantly change with amplitude of the stimulating electric pulses (Fig. 11). To decrease noise, mean data during each term of 0.1 s are traced in Fig ISCUSSION The contractive muscle tube was observed in the second layer from the bottom of the dish. The muscle cells in the bottom

4 layer might tightly be binding on the surface of the culture dish so that the cells cannot slide to generate contraction movement. Fluctuating intensity of the transmitted laser beam includes noise, but can be successfully analyzed on the spectrum. The measurement system enables to detect periodical contraction of muscle tubes at the interval of 0.2 s. The muscle tube might be tetanized with periodical electric stimulation pulses at the interval shorter than 0.2 s. The amplitude of the contraction could not be controlled with amplitude of the electric pulse between 40 V 60 V. Relation between the number of Low intensity High intensity Fig. 12(a): Transmitted light intensity change with contraction of a muscle tube Fig. 12(b): Transmitted light intensity changes with contraction of muscle tubes. Fig. 9: ata digitized from intensity of transmitted laser: relation between intensity (mv) time (ms) at period of 2 s Fig. 10: Spectrum analyzed from data of Fig. 9: relation between intensity frequency (Hz) at period of 2 s. The peak spectrum shows 0.5 Hz. Fig. 12(c): Transmitted light intensity does not change with contraction of muscle tubes. contracting tubes stimulation intensity is not clear, because the observation area is rather small. The sensitivity depends on the position between the laser beam the muscle tube in the present measurement system, because the system can only detect variation of the transmitted laser intensity (Fig.12). 5. CONCLUSION Periodical Contraction of cultured muscle tube has been measured with laser in vitro. The results show that the electric pulses between 0.2 s 2 s. 6. ACKNOWLEGMENT This work was supported by a Grant-in-Aid for Academic Frontier from the Japanese Ministry of Education, Culture, Sports Technology. REFERENCES Fig. 11: Intensity tracings with variation of amplitude (V) of electric pulse. [1] Y. Kawahara, K. Yamaoka, M. Iwata, M. Fujimura, T. Kajiume, T. Magaki, M. Takeda, T. Ide, K. Kataoka, M.

5 Asashima L. Yuge, Novel Electrical Stimulation Sets the Cultured Myoblast Contractile Function to on, Pathobiology, Vol.73, 2006, pp [2] T. Okano T. Matsuda, Tissue Engineered Skeletal Muscle: Preparation of Highly ense, Highly Oriented Hybrid Muscular Tissues, Cell Transplantation, Vol.7, 1998, pp [3] S. Hashimoto, S. Mochizuki, Y. Morita, H. Tsutsui, M. Yoshiura, K. Akazawa, M. Ohsuga, S. Uto, H. Otani T. Fujisato, Environmental esign for Muscle Cell Culture with Magnetic Field, Proc Inaugural IEEE International Conference on igital Ecosystems Technologies (IEEE-EST 2007), 2007, pp [4] 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 K. Yoshinaka, Environmental esign of Muscle Cell Culture for Micro-actuator, Proc. 11th World Multi-conference on Systemics Cybernetics Informatics, Vol.4, pp.30-35, [5] R. G. ennis P. E. Kosnik II, Excitability Isometric Contractile Properties of Mammalian Skeletal Muscle Constructs Engineered in Vitro, In Vitro Cell ev. iol- Animal, Vol.36, 2000, pp [6] T. Okano T. Matsuda, Muscular Tissue Engineering Capillary-Incorporated Hybrid Muscular Tissues in Vivo Tissue Culture, Cell Transplantation, Vol.7, No.5, 1998, pp