Kuniaki Nagamine

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.

Highly Conductive Stretchable and Biocompatible Electrode–Hydrogel Hybrids for Advanced Tissue Engineering

Hydrogel-based, molecular permeable electronic devices are considered to be promising for electrical stimulation and recording of living tissues, either in vivo or in vitro. This study reports the fabrication of the first hydrogel-based devices that remain highly electrically conductive under substantial stretch and bending. Using a simple technique involving a combination of chemical polymerization and electropolymerization of poly (3,4-ethylenedioxythiophene) (PEDOT), a tight bonding of a conductive composite of PEDOT and polyurethane (PU) to an elastic double-network hydrogel is achieved to make fully organic PEDOT/PU-hydrogel hybrids. Their response to repeated bending, mechanical stretching, hydration-dessication cycles, storage in aqueous condition for up to 6 months, and autoclaving is assessed, demonstrating excellent stability, without any mechanical or electrical damage. The hybrids exhibit a high electrical conductivity of up to 120 S cm(-1) at 100% elongation. The adhesion, proliferation, and differentiation of neural and muscle cells cultured on these hybrids are demonstrated, as well as the fabrication of 3D hybrids, advancing the field of tissue engineering with integrated electronics.

Micropatterning contractile C2C12 myotubes embedded in a fibrin gel

Contractile C2C12 myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. The C2C12 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. Biotechnol. Bioeng. 2010;105: 1161–1167.

Electrically induced contraction of C2C12 myotubes cultured on a porous membrane-based substrate with muscle tissue-like stiffness.

A porous membrane-based cell culture device was developed to electrically stimulate a confluent monolayer of C2C12 myotubes. The devices cell culture substrate is a microporous alumina membrane-modified by attaching an atelocollagen membrane on the upperside and a hole-spotted poly(dimethylsiloxane) (PDMS) film on the underside. When electric current is generated between the devices Pt ring electrodes--one of which is placed above the cells and the other below the PDMS layer--the focused current at the PDMS hole can electrically stimulate the cells. C2C12 myoblasts were cultured on the substrate and differentiated into myotubes. When the electrical pulses were applied, myotubes started to contract slightly in and near the hole, and that the continuous stimulation increased both the number of stimuli-responding myotubes and the magnitude of the contraction considerably owing to the underlying atelocollagen membrane with muscle tissue-like stiffness. Also, the generation of contractile myotubes on a wider region of the membrane substrate was possible by applying the electrical pulses through the array of holes in the PDMS film. Using the present system, the glucose uptake by contractile myotubes was examined with fluorescence-labeled glucose, 2-NBDG, which displayed a positive correlation between the contractile activity of myotubes and the uptake of 2-NBDG.

Organic transdermal iontophoresis patch with built-in biofuel cell.

A completely organic iontophoresis patch is reported. A built-in biofuel cell is mounted on the patch that generates transdermal iontophoretic administration of compounds into the skin. The amplitude of transdermal current is tuned by integrating a conducting polymer-based stretchable resistor of predetermined resistance.

Electrophoretic Cell Manipulation and Electrochemical Gene-Function Analysis Based on a Yeast Two-Hybrid System in a Microfluidic Device

A novel microfluidic device with an array of analytical chambers was developed in order to perform single-cell-based gene-function analysis. A series of analytical processes was carried out using the device, including electrophoretic manipulation of single cells and electrochemical measurement of gene function. A poly(dimethylsiloxane) microstructure with a microfluidic channel (150 microm in width, 10 microm in height) and an analytical chamber (100 x 20 x 10 microm (3)) were fabricated and aligned on a glass substrate with an array of Au microelectrodes. Two microelectrodes positioned in the analytical chamber were employed as a working electrode for the electrophoretic manipulation of cells and electrochemical measurements. A yeast strain ( Saccharomyces cerevisiae Y190) carrying the beta-galactosidase reporter gene was used to demonstrate that the device could detect the enzyme. Target cells flowing through the main channel were introduced into the chamber by electrophoresis using the ground electrode laid on the main channel. When the cell was treated with 17beta-estradiol, gene expression was triggered to produce beta-galactosidase, catalyzing the hydrolysis of p-aminophenyl-beta- D-galactopyranoside to form p-aminophenol (PAP). The enzymatically generated PAP was detected by cyclic voltammetry and amperometry at the single-cell level in the chamber of the device. Generator-collector mode amperometry was also applied to amplify the current response originating from gene expression in the trapped single cells. After electrochemical measurement, the trapped cells were easily released from the chamber using electrophoretic force.

Respiration activity of Escherichia coli entrapped in a cone-shaped microwell and cylindrical micropore monitored by scanning electrochemical microscopy (SECM)

The metabolic activity of E. coli cells embedded in collagen gel microstructures in a cone-shaped well and in a cylindrical micropore was investigated using scanning electrochemical microscopy (SECM), based on the oxygen consumption rate and the conversion rate from ferrocyanide to ferricyanide. The analysis of the concentration profiles for oxygen and ferrocyanide afforded the oxygen consumption rate and the ferrocyanide production rate. A comparison indicated that the ferrocyanide production rates were larger than the oxygen consumption rate, and also that the rates observed in the cylindrical micropore were larger than those observed in the cone-shaped well. The ferrocyanide production rate of a single E. coli cell was calculated to be (5.4 +/- 2.6) x 10(-19) mol s(-1), using a cylindrical micropore system.

On-chip electrochemical measurement of β-galactosidase expression using a microbial chip

[small beta]-Galactosidase expression in a small number of Escherichia coli cells has been measured in real time with an electrochemical sensor chip. E. coli cells were embedded using collagen gel within a micropore which was microfabricated onto a chip. The activity of the expressed [small beta]-galactosidase was determined using p-aminophenyl [small beta]-d-galactopyranoside (PAPG) as a substrate.

Porous polymer microneedles with interconnecting microchannels for rapid fluid transport

A porous polymer microneedle array with an average pore diameter of ∼1 μm was fabricated by photopolymerization of an acrylate monomer in the presence of a porogen within a mold. Porosity measurement and water absorption testing revealed that the pores are interconnected throughout the microneedle, which enabled rapid wetting of the microneedle by capillary action. The porosity of the polymer microneedles can be modulated by varying porogen content, and this allowed balancing fast water absorption speed and sufficient mechanical strength to penetrate a skin. The developed porous polymer microneedle array is a potentially versatile tool for rapid fluid transport from and into a body through the skin.

Microfluidic co-cultures of retinal pigment epithelial cells and vascular endothelial cells to investigate choroidal angiogenesis

Angiogenesis plays a critical role in many diseases, including macular degeneration. At present, the pathological mechanisms remain unclear while appropriate models dissecting regulation of angiogenic processes are lacking. We propose an in vitro angiogenesis process and test it by examining the co-culture of human retinal pigmental epithelial cells (ARPE-19) and human umbilical vein endothelial cells (HUVEC) inside a microfluidic device. From characterisation of the APRE-19 monoculture, the tight junction protein (ZO-1) was found on the cells cultured in the microfluidic device but changes in the medium conditions did not affect the integrity of monolayers found in the permeability tests. Vascular endothelial growth factor (VEGF) secretion was elevated under low glucose and hypoxia conditions compared to the control. After confirming the angiogenic ability of HUVEC, the cell-cell interactions were analyzed under lowered glucose medium and chemical hypoxia by exposing ARPE-19 cells to cobalt (II) chloride (CoCl2). Heterotypic interactions between ARPE-19 and HUVEC were observed, but proliferation of HUVEC was hindered once the monolayer of ARPE-19 started breaking down. The above characterisations showed that alterations in glucose concentration and/or oxygen level as induced by chemical hypoxia causes elevations in VEGF produced in ARPE-19 which in turn affected directional growth of HUVEC.