Atsuhito Okonogi

High Throughput Cell Electroporation Array Fabricated by Single-Mask Inclined UV Lithography Exposure and Oxygen Plasma Etching

In this paper, we propose a multiple cell treatment array with high-density arrayed micro-orifice having an individually accessible microchannel and electrodes. More than 10000 micro-orifices with the diameter of 2 mum can be simultaneously fabricated by Single-Mask inclined UV photolithography for embedded network (SIMPLE) process with the oxygen plasma etching. More than 200 living cells immobilized at the arrayed micro-orifices were permeabilized by electroporation and allowed to uptake an foreign impermeant substance.

Open-access and multi-directional electroosmotic flow chip for positioning heterotypic cells

We propose a novel method of cell positioning using electroosmotic flow (EOF) to analyze cell-cell interactions. The EOF chip has an open-to-air configuration, is equipped with four electrodes to induce multi-directional EOF, and allows access of tools for liquid handling and of physical probes for cell measurements. Evaluation of the flow within this chip indicated that it controlled hydrodynamic transport of cells, in terms of both speed and direction. We also evaluated cell viability after EOF application and determined appropriate conditions for cell positioning. Two cells were successively positioned in pocket-like microstructures, one in each micropocket, by controlling the EOF direction. As an experimental demonstration, we observed contact interactions between two individual cells through gap junction channels. The EOF chip should provide ways to elucidate various cell-cell interactions between heterotypic cells.

Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device.

The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.

A perfusable microfluidic device with on-chip total internal reflection fluorescence microscopy (TIRFM) for in situ and real-time monitoring of live cells

A microfluidic device integrated with a Total Internal Reflection (TIR)-based chip for cell observation and analysis was developed. This integrated device enables in situ Total Internal Reflection Fluorescence Microscopy (TIRFM) on adherent cells cultured under continuous medium perfusion. This TIR-based chip, allows TIRFM to be easily performed on cells without the assembly of complicated optical components and cell culture chambers. The integrated device was evaluated by tracking the movement of fluorescent beads and monitoring the location of insulin granules in mouse pancreatic β-cells. This system offers higher signal-to-noise (S/N) ratio than epi-fluorescence microscopy (EPIFM), and comparable image quality to commercial TIRFM systems when imaging insulin granules. We also detected repetitive changes in intracellular Ca2+ concentration in MIN6-m9 cells stimulated with KCl, which demonstrates quick perfusion for cell analysis while maintaining high S/N ratio.

Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells

In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca2+] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells.

Improved hypothermic short-term storage of isolated mouse islets by adding serum to preservation solutions.

Preserving isolated islets at low temperature appears attractive because it can keep islet quantity comparable to freshly isolated islets. In this study, we evaluated the effect of serum as an additive to preservation solutions on islet quality after short-term hypothermic storage. Isolated mouse islets were preserved at 4°C in University of Wisconsin solution (UW) alone, UW with serum, M-Kyoto solution (MK) alone or MK with serum. We then assessed islet quantity, morphology, viability and function in vitro as well as in vivo. Islet quantity after storage in all four solutions was well maintained for up to 120 h. However, islets functioned for different duration; glucose-stimulated insulin release assay revealed that the duration was 72 h when islets were stored in UW with serum and MK with serum, but only 24 h in UW alone, and the islet function disappeared immediately in MK alone. Viability assay confirmed that more than 70% islet cells survived for up to 48 h when islets are preserved in UW with serum and MK with serum, but the viability decreased rapidly in UW alone and MK alone. In in vivo bioassays using 48-h preserved isogeneic islets, all recipient mice restored normal blood glucose concentrations by transplants preserved in UW with serum or MK with serum, whereas 33.3% recipients and no recipient restored diabetes by transplants preserved in UW alone and in MK alone respectively. Adding serum to both UW and MK improves their capability to store isolated islets by maintaining islet functional viability.

Real-time monitoring of Ca 2+ concentration in pancreatic beta cells by a microfluidic device integrated with Total Internal Reflection (TIR)-based chip

We present a microfluidic device integrated with a Total Internal Reflection (TIR)-based chip for cell observation and analysis, which enables Total Internal Reflection Fluorescence Microscopy (TIRFM). By integrating the microfluidic device with optical components, the assembly becomes unnecessary. An evanescent field generated by the TIR-based chip was demonstrated by observation of fluorescent beads and insulin granules in mouse pancreatic beta cells, MIN6-m9. Comparing with epi-fluorescence microscopy (EPIFM), high S/N ratio of 4-fold was realized by the TIR-based chip. To demonstrate the utility of the device integration, we analyzed intracellular Ca2+ concentration change in MIN6-m9 cells in response to KCl stimulation. We found out our integrated device realized quick perfusion for cell analysis with keeping high S/N ratio.

TISSUE-MIMICKING IN VITRO ANALYSIS SYSTEM to exclusively stimulate single cell and detect its physiological reaction

In this paper, we present imaging of the physiological cell reaction of stimulus-responsive cells using a new orifice channel chip named the TISSUE-MIMICKING IN VITRO ANALYSIS SYSTME. The chip enables designated cells treatment using orifice-channels similar to blood vessels, and cell-array like a tissue: cells are along on the blood vessels. These cell handling is an important part in the observation of regulation of cell-functions on stimulus-responsive cells (e.g. pancreas). To study this role, we address to detect cellular stimulation response to designated stimulation using the chip.

Development of a universal integrated micro cell culture system

In this study, we propose an integrated micro cell culture system to easily carry out loading, culturing, observation, and collection of biological cells in a microchannel, and quantitatively evaluated the several factors concerning with the cells in the micro-environment. Especially, we evaluated the effect of the substrate material for the cell culture, and the adhesion and the proliferative properties of the cells collected from the microchannel. It was confirmed that the proposed micro cell culture system is useful for a practical use in cell biology.

Number of kinesin molecules involved in a bead transport measured by microfluidics and mechanical modeling

In contrast to optical tweezers methods, we established a method to evaluate the adhesion force between a bead carried by multiple kinesins and a microtubule. The method also enables us to estimate the number of kinesins involved in the bead transport. Torque Γ and drag force FD applied by the shearing flow were derived theoretically by the flow rate necessary to break kinesin-microtubule bindings in the bead detachment assay. Based on mechanical modeling, the adhesion force, FAdh, was calculated as 362.9 pN without ATP and 31.3 pN with 1 mM ATP. Furthermore, we found out 5.5–11.9 kinesins are cooperatively working to carry the bead, which is consistent with other results. Our method is advantageous in analyzing multi-kinesin transport system reconstructed in microfluidic systems.