Haruka Hino

Are Micro Back Markers on Thin Film of Scaffold Effective to Evaluate Contractive Movement of Myotube

The scaffold of the transparent film with micro markers has been designed to evaluate the contractive movement of myotube under the electric pulse stimulations in vitro. The scaffold of a thin film (6 μm of thickness) of polydimethylsiloxane was made with ten million micro markers: 4 μm of diameter, 2 μm of height, and 30 μm of interval. Ten million markers were made on the backside of the scaffold by the photolithography technique. C2C12 (mouse myoblast cell line) was seeded on the film at the counter surface to the markers at the density of 5000 cells/cm2. The cells were cultured for 12 days in the medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 310 K with 5% of CO2 content. The electric pulses (1 s of pulse cycle, 1 ms of pulse width) were applied between platinum electrodes dipped in the medium. The myoblasts were able to be cultured on the film to be differentiated into myotubes. The thin film with micro markers was successfully made of polydimethylsiloxane, and had enough transparency for observation of myotubes by the microscope. The myotube was contracted synchronously with the stimulation of electric pulses. The contractile movement was able to be analyzed by the repetitive displacement of the length between backside micro markers of the film. The micro backside markers on thin film of scaffold are effective to evaluate contractive movement of the myotube.

How Does Cell Deform Through Micro Slit Made by Photolithography Technique

Several slits sort cell according to the deformability in vivo. A micro slit (0.87 mm of width, 0.010 mm height) was newly designed between a micro ridge on a transparent polydimethylsiloxane plate and micro ridges on a borosilicate glass plate. These ridges made by photolithography technique make contact each other in the perpendicular position to make the slit between the ridges. A one-way flow system was designed to observe each cell passing through the slit in vitro. Four kinds of cells were used in the test: C2C12 (mouse myoblast cell), HUVEC (human umbilical vein endothelial cell), Hepa1-6 (mouse hepatoma cell), and Neuro-2a (mouse neural crest-derived cell). The suspension of each kind of cells was injected to the slits. The deformation of each cell passing through the slit was observed with an inverted phase-contrast microscope. At the microscopic images, the outline of each cell was traced, and the area (S) was calculated. The deformation ratio was calculated as the ratio (S2/S1) of the projected area of each cell before the slit (S1) and that in the slit (S2). The velocity of the cell passing through the slit was calculated by the trace at the microscopic movie. The experimental results show that each cell deforms to the flat circular disk and passes through the micro slit. Hepa1-6 is flattened with the increase of the passing velocity, and HUVEC is elongated along the flow. The designed slit between micro ridges is effective to evaluate the deformability of cells.