Biomedical subjects

Matsuhiko Nishizawa

Publications and source records attributed to Matsuhiko Nishizawa.

16 recordsLinked to original sources

PubMed2006

Electrodeposition of anchored polypyrrole film on microelectrodes and stimulation of cultured cardiac myocytes.

The electrically conducting polymer polypyrrole (PPy) was electrochemically deposited onto Pt microelectrodes on a polyimide (PI) substrate. Pre-modification of the PI surface with a self-assembled monolayer of octadecyltrichlorosilane-induced anisotropic lateral growth of PPy along the PI surface and enhanced adhesive strength of the PPy film. The lateral growth of PPy film around the electrode anchored the whole film to the substrate. External stimulation of cultured cardiac myocytes was carried out using the PPy-coated microelectrode. The myocytes on the microelectrode substrate were electrically conjugated to form a sheet, and showed synchronized beating upon stimulation. The threshold charge for effective stimulation of a 0.8 cm(2) sheet of myocytes was around 0.2 microC, roughly corresponding to a membrane depolarization of 250 mV.

Animals↗

PubMed2006

Patterning cellular motility using an electrochemical technique and a geometrically confined environment.

We describe herein a method for controlling the pattern of permissible cell migration and proliferation on a substrate in time and space. Using this method, a confluent monolayer of cells that is confined within a defined region is released into a neighboring region. Incorporated into the method is an electrochemical technique that uses a scanning microelectrode to draw regions on the surface of the system that thereafter can support cell migration and growth. The supporting glass substrate is patterned with regions of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer that are not affected by the electrochemical treatment and also robustly resist cellular overgrowth as well as regions that can be individually switched when electrochemically treated from cell repellent to cell adhering. It is therefore possible to strictly define the areas into which cells can migrate. We found that HeLa cells migrate more rapidly as the width of cell-adhering lanes increases until a width of ca. 50 microm is reached, at which point the migration rate is roughly constant. We also designed a drug assay using our cell migration technique. The technique allows for cell migration only into defined region(s) and therefore may become an important tool for evaluating the biological activity of potential drugs because drug activity and cell motility often directly correlate.

Journal Article↗

PubMed2006

On-demand patterning of protein matrixes inside a microfluidic device.

On-demand immobilization of proteins at specific locations in a microfluidic device would advance many types of bioassays. We describe a strategy to create a patterned surface within a microfluidic channel by electrochemical means, which enables site-specific immobilization of protein matrixes and cells under physiological conditions, even after the device is fully assembled. By locally generating hypobromous acid at a microelectrode in the microchannel, the heparin-coated channel surface rapidly switches from antibiofouling to protein-adhering. Since this transformation allows compartmentalizing of multiple types of antibodies into distinct regions throughout the single microchannel, simultaneous assay of two kinds of complementary proteins was possible. This patterning procedure can be applied to conventional microfluidic devices since it requires only some electrodes and a voltage source (1.7 V, DC).

Animals↗

PubMed2005

Patterning the surface cytophobicity of an albumin-physisorbed substrate by electrochemical means.

Spatiotemporal control of surface properties under physiological conditions such as those found in culture media is an important technique in fundamental cell biology, tissue engineering, and cell-based bioelectronics. To this end, we have developed a mild, wet cellular micropatterning technique. The principle of the technique is based on the fact that the cell-repellant property of the albumin-coated substrate rapidly switches to cell-adhesive upon exposure to the reactive oxidizing agent, electrochemically generated hypobromous acid. Herein, we report the effect of the hypobromous acid on serum albumin physisorbed on a hydrophobic substrate. It was found that albumin molecules detach from the substrate by application of the oxidizing agent, resulting in exposure of the underlying hydrophobic surface to the liquid phase. The adsorption of extracellular matrix proteins such as fibronectin onto the hydrophobic surface induces cell adhesion and growth.

Adsorption↗

PubMed2005

Multi-channel 3-D cell culture device integrated on a silicon chip for anticancer drug sensitivity test.

A novel three-dimensional cell culture system was constructed with an array of cell panels (4 x 5) in a silicon chip, together with multi-channel drug containers. Human breast cancer (MCF-7) cells were embedded in a collagen-gel matrix and entrapped in a pyramidal-shaped silicon hole. Each cell panel can be isolated by a channel composed of a microfluid part and a reservoir. A cell panel was exposed to 200 mm KCN for 2 days to demonstrate that each cell panel could be independently evaluated under various stimulation conditions. Based on the cellular respiration activity, the proliferation behavior was continuously monitored on the silicon-based cell array for 5 days using scanning electrochemical microscopy (SECM). The cells entrapped in the device (3-D culture) proliferated normally, and the proliferation rate was lower than that of cells grown in a monolayer cell culture (2-D culture). The effects of three anticancer drugs measured simultaneously on the cell chip were in good agreement with those obtained by a conventional colorimetric assay. Our results suggest that the silicon-based device for 3D culture is appropriate for a chemosensitivity assay involving multi-chemical stimulation.

Antineoplastic Agents↗

PubMed2004

In situ control of cellular growth and migration on substrates using microelectrodes.

We describe an electrochemical method to direct the growth and migration of mammalian cells on a substrate during cultivation in situ. Exposing the albumin-coated substrate to an oxidizing agent, hypobromous acid, electrochemically generated at the tip of the scanning microelectrode, locally switched the substrate from cytophobic to cell-adhesive. This transformation generated the formation of cellular micropatterns. Since the concentration of the oxidizing agent required for the surface processing did not cause significant damage to the cell cultures, we were able to direct in situ cellular proliferation and migration by drawing adhesive micropatterns over the preexisting cellular pattern.

Journal Article↗

PubMed2004

Proliferation assay on a silicon chip applicable for tumors extirpated from mammalians.

We describe a novel anticancer drug sensitivity assay on a silicon chip applicable for tumors extirpated from in vivo mammalians. Human promyelocytic leukemia (HL-60) cells were subcutaneously (s.c.) inoculated in SCID mice, then removed 31 days after the inoculation. The cells were embedded in a small volume (18 nL) of a collagen-gel matrix on a pyramid-shaped silicon microstructure for further cultivation. The respiration activity of the cells on the chip was measured by scanning electrochemical microscopy (SECM). The proliferation behavior was continuously monitored for 6 days. It seemed that the proliferation rate of the cells removed from the mice was lower than that cultured in a flask and conformed to that in mice. The effects of cisplatin (CDDP) and etoposide (VP-16) on the HL-60 cultured in vivo were in good agreement with those obtained by a conventional colorimetric assay. Our results suggest that the SECM-based assay is appropriate for biopsy specimens in a relatively short-time evaluation.

Animals↗

PubMed2004

Microelectrochemical approach to induce local cell adhesion and growth on substrates.

The nature of an albumin-coated substrate that blocks protein adsorption and cell adhesion was rapidly switched to cell-adhesive by exposure to an oxidizing agent such as HBrO. This finding has enabled cellular pattern drawing even on a single-cell level by closely scanning a microelectrode above the substrate and electrochemically producing the agent at the tip of the electrode. The present microelectrochemical cell patterning is applicable even for a previously cell-patterned substrate and for a grooved substrate. These unique technical features will have impacts on a variety of cell-based studies that require the analysis of heterotypic cell-cell interactions and cellular arrangement on an uneven surface such as semiconductor devices.

Bromates↗

PubMed2003

Monitoring the cellular activity of a cultured single cell by scanning electrochemical microscopy (SECM). A comparison with fluorescence viability monitoring.

The respiratory activities of cultured HeLa cells were monitored at a single cell level using scanning electrochemical microscopy (SECM) that produces images of the localized distribution of oxygen around the cell. The change in the cellular activity was traced after exposures to KCN, ethyl alcohol and the antibiotic drug, Antimycin A. The results were compared with those from the conventional fluorescence monitoring using Calcein-AM that is sensitive to deformation of the cell membrane. The SECM-based measurement follows the decrease in the cellular activity upon exposure to KCN and Antimycin A more rapidly than the fluorescence-based measurements, demonstrating that SECM is suitable for studying the cellular influence of respiration inhibitors.

Antimycin A↗

PubMed2003

Fabrication of microbial chip using collagen gel microstructure.

A microbial chip was fabricated by filling the micropores on a glass substrate with collagen-embedded Escherichia coli(E. coli) cells, and characterized by scanning electrochemical microscopy (SECM) in a solution containing ferricyanide. The activity of the E. coli cells in the collagen gel microstructure was imaged and characterized with SECM by mapping the localized concentration of ferrocyanide produced by the respiration of the cells. The SECM-based activity measurement detected as low as approximately 100 E. coli cells. Furthermore, the optical-microscopic observation indicated that the E. coli cells on the chip proliferated during the incubation. The sequential SECM measurements were performed for the same E. coli chip to obtain the microbial growth curve for a small number of microorganisms.

Collagen↗

PubMed2003

Localized chemical stimulation to micropatterned cells using multiple laminar fluid flows.

Micropatterns of the electrically conjugating cardiomyocytes were prepared on a single cell level by microcontact printing (microCP), and the localized chemical stimulations were applied to the cellular pattern using multiple laminar flows. The locally delivered 1-octanol inactivated part of the myocyte patterns, while the other areas retained the activity showing spontaneous and synchronous pulsatility. Since both the cells and flows were well defined as micropatterns and each integrated on a chip, the obtained results simply demonstrate the cellular responses in a single-cell network.

1-Octanol↗

PubMed2003

Scanning electrochemical microscopy-based drug sensitivity test for a cell culture integrated in silicon microstructures.

The respiratory activity of collagen-embedded living cells was imaged by scanning electrochemical microscopy (SECM) with the objective to study anticancer drug sensitivity. Two kinds of cancer cells, the human erythroleukemia cell line (K562) and its adriamycin-resistant subline (K562/ADM), were immobilized at the array of microholes micromachined on a silicon wafer for comparative characterization of their sensitivity to the anticancer drug, ADM. The results obtained by the SECM method showed correspondence to a conventional colorimetric assay (SDI assay). Furthermore, since the SECM assay is based on the noninvasive measurement of the respiration activity, continuous monitoring of a dose response was possible.

Dose-Response Relationship, Drug↗

PubMed2003

Intracellular Ca2+ imaging for micropatterned cardiac myocytes.

The patterning of cardiac myocytes on a micron scale ( approximately 5 microm) was achieved by microcontact printing of fibronectin onto a hydrophobically pretreated glass substrate. The patterned cardiac myocytes conjugated with each other by forming a gap junction, as judged from the synchronized Ca(2+) transition over the pattern, and thus simultaneously contracted. The dynamic change of the Ca(2+) concentration within the patterned tissue was analyzed quantitatively during successive contraction and relaxation using a Nipkow-type high-speed confocal microscope.

Calcium↗

PubMed2003

Pharmacological characterization of micropatterned cardiac myocytes.

The cardiac myocytes patterned on a single cell level were prepared by microcontact printing, and the response to chemical stimuli was studied using confocal fluorescent Ca(2+) imaging. The patterned myocytes were found to conjugate by forming gap junction. It was confirmed for the patterned myocytes that gap junction communication was reversibly inhibited by 1-octanol, but activated by caffeine. Localized stimulation with chemicals was also attempted using a microinjection system for the myocyte patterns formed by single cell alignment. This research was carried out with the objective of developing a bioassay system based on a cellular network.

1-Octanol↗

PubMed2003

Imaging of immobilized enzyme spots by scanning chemiluminescence microscopy with electrophoretic injection.

Scanning chemiluminescence microscopy (SCLM) with electrophoretic injection was developed and applied to visualize enzyme reactions localized in an enzyme microspot. The SCLM uses a tapered glass capillary as a probe for injecting a small amount of luminol onto the substrate to generate localized chemiluminescence. The electrophoretic injection by application of a constant current between the inside and outside of the capillary enabled the continuous and controllable injection of a minute quantity of luminol in the range of 0.1 pmol/s. The image of enzyme activity in a monolayer spot of horseradish peroxidase was obtained by using the electrophoretic injection-based SCLM system.

Electrophoresis↗

PubMed2003

Imaging of enzyme activity by scanning electrochemical microscope equipped with a feedback control for substrate-probe distance.

The enzymatic activity of diaphorase (Dp) immobilized on a solid substrate was characterized using a scanning electrochemical microscope (SECM) with shear force feedback to control the substrate-probe distance. The shear force between the substrate and the probe was monitored with a tuning fork-type quartz crystal and used as the feedback control to set the microelectrode probe close to the substrate surface. The sensitivity and the contrast of the SECM image were improved in the constant distance mode (distance, 50 nm) with the shear force feedback compared to the image in the constant height mode without the feedback. By using this system, the SECM and topographic images of the immobilized diaphorase were simultaneously measured. The microelectrode tip used in this study was ground aslant like a syringe needle in order to obtain the shaper topographic images. This shape was also effective for avoiding the interference during the diffusion of the enzyme substrates.

Electrochemistry↗