Session: 06-03-02 Biologically Enabled Microfluidics and Biomicrofluidics

Paper Number: 65639

Start Time: August 11th, 10:50 AM

65639 - Hysteresis Effect of Tangential Force Field With Centrifuge on Myoblast: Cultured on Striped Pattern of Micro Ridge for Direction Control 

The relationship between strengthen of the muscle tissue and the gravitational force has been highlighted in the study field of the space technology. In the environment of microgravity, the muscle tissue degenerates. Hypergravity environment, on the other hand, strengthens the muscle tissue. The behavior of biological cells depends on the force field both in vivo and in vitro. Cells make orientation to make a tissue. Endothelial cells, for example, make orientation parallel to the flow direction on the inner surface of the blood vessel wall. To elucidate the control method of cell orientation would be useful for the engineered tissue formation in the field of regenerative medicine. The recent cell culture technique with the time-lapse images enables tracking observation of the behavior of each cell in vitro. A biological cell adheres on the scaffold and shows several active behaviors: migration, deformation, proliferation, and differentiation. The cell moves by itself. The cell can deform to minimize the intra force of itself.

In the present study, C2C12 (mouse myoblast cell line) has been used. Both the migration and the deformation of each cell has been restricted by the wall shear stress higher than 3 Pa exposed to the flow in vitro. Deformability of the floating single cell has been observed through the slit in the micro flow channel in vitro. Even if myoblasts are cultured in a continuous normal centrifugal force field in an incubator, myoblasts differentiate into myotubes to make a layer of myotubes. The excess normal force makes thicker myotubes. The tangential force, on the other hand, can affect orientation of each cell. The effect depends on the initial alignment of the cell. While the strong stimulation above the threshold damages the cell, the stimulation below the threshold remains in the cell as a memory for the response in the next step. The memory can be reset after division of the cell. The longitudinal axis of C2C12 tends to align to the direction of the tangential force. The axis tilts to the perpendicular direction, on the other hand, after stopping of the tangential force. Although a single cell makes alignment at random on the scaffold, each cell tends to align to the longitudinal direction of micro ridges higher than 0.7 µm on the scaffold surface.

The effect of hysteresis of the tangential force field at the surface of the scaffold on the behavior of myoblast has been studied in vitro. To examine the effect of the direction of the tangential force field on the cell at the specific alignment, the direction of each cell was controlled by the topography on the surface of the scaffold. The micro striped ridge-pattern (0.7 μm height, 3 μm width, and 3 μm interval) was made on the surface of the scaffold plate by the photolithography technique. Variation was made on the angle between the longitudinal direction of the ridge and the direction of the tangential force: 0, 45, and 90 degrees. To apply the tangential force field to the cells, the scaffold plate was set in the tube in a conventional centrifugal machine placed in an incubator. During cultivation in the medium fluid, the tangential force associated with the centrifugal force is generated by the difference of the density between the cell and the medium (< 6 G). After the centrifugation for 5 hours, the behavior of each cell at the time-lapse microscope images was traced for 24 hours to analyze the behavior of each cell related to the direction of the tangential force field. The experimental results show that the tendency of cells to make alignment to the longitudinal direction of the striped pattern is strengthened by the hysteresis of the exposure to the tangential force field. The tendency is remarkable, when the stripe pattern is parallel to the tangential force. The results would contribute to select the mechanical environment to make the engineered muscle tissue.

Presenting Author: Shigehiro Hashimoto Kogakuin University

Authors:

Shigehiro Hashimoto Kogakuin University