We have developed gel sheet-supported C(2)C(12) myotube micropatterns and combined them with a microelectrode array chip to afford a skeletal muscle cell-based bioassay system. Myotube line patterns cultured on a glass substrate were transferred with 100% efficiency to the surface of fibrin gel sheets. The contractile behavior of each myotube line pattern on the gel was individually controlled by localized electrical stimulation using microelectrode arrays that had been previously modified with electropolymerized poly(3,4-ethylenedioxythiophene) (PEDOT). We successfully demonstrated fluorescent imaging of the contraction-induced translocation of the glucose transporter, GLUT4, from intracellular vesicles to the plasma membrane of the myotubes. This device is applicable for the bioassay of contraction-induced metabolic alterations in a skeletal muscle cell.

In this study, we prepared injectable collagen microspheres for the sustained delivery of recombinant human vascular endothelial growth factor (rhVEGF) for tissue engineering. Collagen solution was formed into microspheres under a water-in-oil emulsion condition, followed by crosslinking with water-soluble carbodiimide. Various sizes of collagen microspheres in the range of 1-30 mum diameters could be obtained by controlling the surfactant concentration and rotating speed of the emulsified mixture. Particle size proportionally decreased with increasing the rotating speed (1.8 mum per 100 rpm increase in the range of 300-1,200 rpm) and surfactant concentration (3.1 mum per 0.1% increase in the range of 0.1-0.5%). The collagen microspheres showed a slight positive charge of 8.86 and 3.15 mV in phosphate-buffered saline and culture medium, respectively. Release study showed the sustained release of rhVEGF for 4 weeks. Released rhVEGF was able to induce capillary formation of human umbilical vein endothelial cells, indicating the maintenance of rhVEGF bioactivity after release. In conclusion, the results suggest that the collagen microspheres have potential for sustained release of rhVEGF.

We investigated the interactions between HeLa cells and human umbilical vein endothelial cells (HUVECs) by monitoring their movements in a controllable coculture system. Two complementary, detachable, cell-substrates, one of polystyrene (PS) and the other of poly(dimethylsiloxane) (PDMS), were fabricated by replica molding. Coculturing was started by mechanically assembling two complementary substrates. One substrate was covered with a confluent layer of HeLa cells and its complement covered with confluent HUVECs. Using this coculture system as a tumor/endothelium model, we found that the HeLa cells migrated towards the HUVECs, while, simultaneously, the HUVECs retreated and that both types of cells migrated approximately twice as rapidly (two hundred microns per twenty-four hours) as they did alone. Additionally, when direct contact between the two cell types was prevented, the HUVECs initially migrated towards the HeLa cells and then retreated. The characteristics of the cell movements, i.e. direction and speed, probably are consequences of cell-cell signaling, with such signals possibly important during tumor cell intra- and extravasation.

Contractile C(2)C(12) myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. The C(2)C(12) myotube/fibrin gel system was prepared by transferring a myotube monolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. To endow the myotubes with contractile activity, a series of electrical pulses was applied through a pair of carbon electrodes placed at either side of a fibrin gel separately. The frequency and magnitude of myotube contraction were functions of the pulse frequency and duration, respectively. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (1 week) than myotubes adhered on a conventional culture dish.