Session: 13-04-02: Applications of Micro and Nano Systems in Medicine and Biology II

Paper Number: 112503

112503 - Evaluation of Myotubes Orientation Cultured on Scaffold Film by Micromarkers Matrix 

Recently, engineered tissues have been studied based on cell culture technology. Cells attach to the surface as a scaffold and perform various activities in vitro as well as in vivo: migration, deformation, division, and differentiation. Myoblasts, which are the source of skeletal muscle tissue formation, proliferate and fuse to form myotubes (differentiation).

Myotubes gather to form muscle fibers. Muscle tissue consists of muscle fibers. Skeletal muscles connect to the nervous system and contract via electrical signals. The muscle contraction movement is realized by the direction and timing of contraction of each muscle fiber. Without control as a whole system, the activity of each cell is random. Every parameter is random: the direction of cells, the degree of fusion, the size and direction of myotubes.

Myotubes formed on the surface of the scaffold contract by applying electrical stimulation to the culture medium in vitro. In a single contraction, motion occurs between two points at opposite phase timings. Both the amplitude and timing of each movement depend on the position. Under an optical microscope, it is not easy to track the movement of a particular position within the myotubes. Appropriate markers are needed.

In this study, micro-protrusions were designed as micro-markers on a thin film. The shape and spacing of each protrusion in the matrix pattern were determined with reference to the size of myotubes. Each protrusion was hemispherical (diameter 4 µm, height 2 µm). The pitch between adjacent protrusions was 30 µm.

The micro-protrusions were created integrally with a polydimethylsiloxane (PDMS) film. The thickness of PDMS film was 6.4 µm. The thickness of PDMS film scaffold was selected for myotubes to occur repetitive contraction by electric stimulation via the medium. The matrix pattern of protrusions was created in a 3 mm × 3 mm square area in the center of the film by photolithography technology. The film was sandwiched and supported between a pair of PDMS donut rings (10 mm inner diameter).

Mouse myoblasts were seeded on the surface of the opposite side of thin film and incubated for 10 days to differentiate into myotubes. Periodic electrical pulses (amplitude 2 V, pulse period 0.5 s, pulse width 1 ms) were applied between the electrodes of the titanium wire immersed in the medium. The movements of the myotube synchronized with the electric pulses were observed under the microscope. Synchronized movements of the matrix pattern micromarkers were recorded with a movie camera. A still image of 30 frames per second was cut out from the movie, and the centroid of each marker was tracked over the cycle of electrical stimulation.

The position of each marker shows cyclic movements. At each tracing, the amplitude and timing were measured. Several local areas were selected. Each area contains 5 × 5 markers. In the area, the movement angles (α) were calculated by the amplitude and timing. The major axis direction (θ) of each myotube was measured in the area on the microscopic image. The mean values of α and θ in the area were compared.

The movement of the myotube consists of contraction and relaxation (restoration). A pair of muscles contract alternately to make repetitive movements in vivo. Appropriate repulsive force is required for the scaffolding in vitro. In this study, a thin film supported by the ring was selected as a scaffold for myotubes. The method is useful for observing the distribution of contractions.

In the myotubes layer formed in vitro, the randomness of the myotubes distribution complicates the movement measurement. On the other hand, it is possible to evaluate the distribution of myotubes contraction by the local measurement. The evaluation leads to an orientation assessment of the myotubes tissue formed in vitro. The technology may contribute to the field of regenerative medicine.

Presenting Author: Shigehiro Hashimoto Kogakuin University

Presenting Author Biography: 1972-1975 Tokyo Gakugei University Senior High School, Tokyo, Japan.
1975-1979 Bachelor of Engineering, Department of Mechanical Physics, School of Engineering, Tokyo Institute of Technology, Tokyo, Japan.
1977 Internship Student (from July to August), Research Center for Artificial Heart, Free University in Berlin, Berlin, Germany.
1979-1981 Master of Engineering, Mechanical Engineering, Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo, Japan.
1987 Doctor of Medicine at Kitasato University, Sagamihara, Japan.
1990 Doctor of Engineering at Tokyo Institute of Technology, Tokyo, Japan.

Professional experience:
1981-1989 Research Associate, School of Medicine, Kitasato University, Sagamihara, Japan.
1989-1994 Assistant Professor, School of Medicine, Kitasato University, Sagamihara, Japan.
1994-2001 Associate Professor, Department of Electronics, School of Engineering, Osaka Institute of Technology, Osaka, Japan.
2001-2004 Professor, Department of Electronics, School of Engineering, Osaka Institute of Technology, Osaka, Japan.
2004-2006 Professor, Department of Electronics, Information and Communication Engineering, School of Engineering, Osaka Institute of Technology, Osaka, Japan.
2004-2005 Chair, Department of Electronics, Information and Communication Engineering, School of Engineering, Osaka Institute of Technology, Osaka, Japan.
2005-2009 Director, Medical Engineering Research Center, Osaka Institute of Technology, Osaka, Japan.
2005-2006 Coordinator, Department of Biomedical Engineering, School of Engineering, Osaka Institute of Technology, Osaka, Japan.
2006-2011 Professor, Department of Biomedical Engineering, School of Engineering, Osaka Institute of Technology, Osaka, Japan.
2011- Professor, Department of Mechanical Engineering, School of Engineering, Kogakuin University, Tokyo, Japan.
2012-2018 Associate to President and Dean of Admissions Center, Kogakuin University, Tokyo, Japan.
2017-2020 Councilor, Kogakuin University, Tokyo, Japan.
2018-2021 Dean, School of Engineering, Kogakuin University, Tokyo, Japan.
2020-2021 Chair, Systems Design, Graduate School of Engineering, Kogakuin University, Tokyo, Japan.

2021-2023 The Society of Life Support Engineering, Japan (President).
2022-2025 Associate Editor ASME Journal of Engineering and Science in Medical Diagnostics and Therapy

Authors:

Shigehiro Hashimoto Kogakuin University
Shusei Sakai Kogakuin University
Shunsuke Saito Kogakuin University