Soichiro Sekine

Conducting Polymer Electrodes Printed on Hydrogel

We report herein the micropatterning of poly(3,4-ethylenedioxythiophene) (PEDOT) on a hydrogel, agarose, to provide a fully organic, moist, and flexible electrode. The PEDOT/agarose electrodes were prepared through two electrochemical processes: electropolymerization of PEDOT into the hydrogel and electrochemical-actuation-assisted peeling. We also present a typical application of the PEDOT/agarose electrode to the cultivation of contractile myotubes.

Electrodes Combined with an Agarose Stamp for Addressable Micropatterning

We have combined a topographically patterned agarose microstamp with an electrode substrate to develop a novel printing device that internally contains an electrochemical system for a controlled supply of reactive ink to the stamp surface. The 10 wt % agarose gel containing 0.1 M PBS + 25 mM KBr showed suitable elasticity for forming stamps and served as the electrolytic medium for the electrochemical oxidation of Br(-) to generate HBrO. The electrode substrate patched with an agarose stamp having 50-microm-high bumps was used for the spatially confined detachment of heparin/polyethyleneimine precoated on glass substrates, followed by micropatterned adsorption of fibronectin. Using the microelectrode array, the addressable micropatterning of protein by the controlled delivery of HBrO to each bump was demonstrated.

Metabolic Assay System for Micropatterned Contractile Myotubes

In vitro bioassay system incorporating skeletal muscle cells is required to reveal the complex mechanisms involved in the development and maintenance of type2 diabetes because type2 diabetes is closely associated with defection of glucose uptake in skeletal muscle cells. An assay system, currently available, that is used to monitor skeletal muscle cell contraction and activity consists of myotube monolayer cultured on substrates with a pair of electrodes for stimulation. By applying electric pulse stimulation, sarcomere assembly is accelerated and allows for cell contraction. However, the contracting myotubes were difficult to maintain their structure for a long period of time because they readily detached from the substrate within a few days. In this study, we prepared contractile C2C12 myotube line patterns embedded in a fibrin gel to afford a physiologically relevant and stable bioassay system. The C2C12 myotube/fibrin gel system was prepared by transferring a myotube m onolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. The frequency and magnitude of myotubes contraction were functions of the pulse frequency and duration, respectively. The patterned C2C12 myotubes, supported by the elastic fibrin gel, showed larger contractile displacement than did a myotube monolayer that was attached on a culture dish. The myotubes/fibrin gel system also maintained the line pattern and contractile activity for a longer period of time (one week) than did the myotubes of the dish system. This C2C12 myotube/fibrin gel sheet was easy to handle, allowing to be attached and be aligned onto the paired microelectrode arrays chip (Figure 3(A), (B)). Figure 3 (C) shows the time course of contractile displacements of myotube line patterns. (1)-(3) in Fig. 3(C) represent the myotube line patterns shown in Fig.3(B). All the myotube line patterns were electrically stimulated at first followed by site-specific electricalstimulation against myotube (2), exhibiting independent contractile behavior of each myotube line pattern on the microelectrode arrays. This device would enable highly-accurate bioassay of myotubes by patterning test and control myotubes with different contractile activity next to each other. Now, we are investigating contraction-mediated translocation of the GLUT4 glucose transporter protein in myotubes using this system. On the other hand, we have developed a novel technique for printing a micropatterned conducting polymer film on a hydrogel. Pt microelectrode on a glass plate was used as a master for electrodeposition of conducting polymer, PEDOT, into a fibrin gel sheet. After the electrodeposition, the gel sheet with micropatterned PEDOT can be peeled from the substrate (Fig. 2A). This flexible gel-based electrode was used for effective stimulation of the C2C12 myotubes by binding the two gel sheets: one contains C2C12 micropatterns; the other contains PEDOT micropatterns (Fig. 2B,C).

Electrochemical In-Situ Micropatterning of Cells and Polymers

We report two novel techniques “Electrochemical Bio-Lithography” that enables in-situ cellular micropatterning, and “Ultra Anisotropic Electrodeposition” for making micropatterns of conducting polymers. The former technique for cellular micropatterning was combined with dielectrophoresis (DEP) and AFM, and we have realized the in-situ spatiotemporal cellular micropatterning in various environments. On the other hand, we have found the micropatterned hydrophobic area around electrode serves as a template for in-situ circuit formation with conducting polymers. Importantly, since the electropolymerization can be conducted without significant damage to the existing cell cultures, these two techniques can be combined to form hybrid micropatterns of cells and conducting polymers.

Generation of Patterned Cell Co-Cultures inside Tubular Structure Using Electrochemical Biolithography and Electrostatic Assembly

We report a method for producing patterned cell co-cultures inside silicone tubing. A platinum needle microelectrode was inserted through the wall of the tubing and an oxidizing agent electrochemically generated at the inserted electrode. This agent caused local detachment of the anti-biofouling heparin layer from the inner surface of the tubing. The cell-adhesive protein fibronectin selectively adsorbed onto the newly exposed surface, making it possible to initiate a localized cell culture. The electrode could be readily set in place without breaking the tubular structure and, importantly, almost no culture solution leaked from the electrode insertion site after the electrode was removed. Ionic adsorption of poly-L-lysine at the tubular region retaining a heparin coating was used to switch the heparin surface from cell-repellent to cell-adhesive, thereby facilitating the adhesion of a second cell type. The combination of the electrode-based technique with electrostatic deposition enabled the formation of patterned co-cultures within the semi-closed tubular structure. The controlled co-cultures inside the elastic tubing should be of value for cell-cell interaction studies following application of chemical or mechanical stimuli and for tissue engineering-based bioreactors.

Generation of Patterned Cell Co-Cultures in Silicone Tubing Using a Microelectrode Technique and Electrostatic Assembly

We report a method for producing patterned cell adhesion inside silicone tubing. A platinum needle microelectrode was inserted through the wall of the tubing and an oxidizing agent electrochemically generated at the inserted electrode. This agent caused local detachment of the anti-biofouling heparin layer from the inner surface of the tubing. The cell-adhesive protein fibronectin selectively adsorbed onto the newly exposed surface, making it possible to initiate a localized cell culture. The electrode could be readily set in place without breaking the tubular structure and, importantly, almost no culture solution leaked from the electrode insertion site after the electrode was removed. Ionic adsorption of poly-L-lysine at the tubular region retaining a heparin coating was used to switch the heparin surface from cell-repellent to cell-adhesive, thereby facilitating the adhesion of a second cell type. The combination of the electrode-based technique with electrostatic deposition enabled the formation of patterned co-cultures within the semi-closed tubular structure. The controlled co-cultures inside the elastic tubing should be of value for cell-cell interaction studies following application of chemical or mechanical stimuli and for tissue engineering-based bioreactors.