小沢 文智

Abstract Redox mediators (RMs) suppress the charging overpotential to enhance the cycle performance of lithium-air batteries (LABs), but inappropriate RM incorporation can adversely shorten cycle life. In this study, three typical organic RMs; tetrathiafulvalene (TTF), 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), and 10-methylphenothiazine (MPT), were incorporated into the air-electrode (AE) of the LAB (RM-on-AE), rather than dissolving them in the electrolyte (RM-in-EL), to maximize the RM effect throughout the cycle life. The discharge/charge cycle test confirmed that the cells with RM-on-AE prevented the reductive decomposition of RM with the lithium anode, deriving the RM effect for a longer cycle life than the cells with RM-in-EL. The measurement of AE deposits revealed that the TTF- and TEMPO-on-AE cells failed to generate a quantitative amount of Li2O2 discharge product. In contrast, the MPT-on-AE provided a 96% yield of Li2O2 after the first discharge because of the reductive tolerance of the MPT as organic RM. The quantitative analysis also revealed an accumulation of Li2CO3 on the AEs, along with the generation of carboxylate, as the side products of irrelevant battery reactions. This study provides a practical methodology for selecting RMs and their incorporation for developing long-life LABs.

Four ethers were compared as solvents of lithium naphthalenide (Li-NTL) solutions to pre-dope Li into Si electrodes. The solvents of the Li-NTL solutions affected the stability and equilibrium potential (V (eq)). X-ray diffraction, thermodynamic characterization and ultraviolet-visible (UV-vis) spectroscopy were used to clarify the effects of the solvation structure, the lowest unoccupied molecular orbital (LUMO) energy of the solvent molecule and the ion pair structure between Li+ ions and naphthalenide radical anions ([NTL](center dot-)) on doping capacity. A Li-NTL solution having a low V (eq) and sufficient stability under potentials as low as that of Li metal was found to provide the highest pre-doping capacity. In particular, a 2-methyltetrahydrofuran (MeTHF) solution exhibiting the lowest V (eq) showed a pre-doping capacity as high as 3250 mAh g(-1) after 24 h. UV-vis spectra and Walden plots indicated that a Li-NTL solution using MeTHF provided less dissociation than a tetrahydrofuran (THF) solution. The doping capacity is evidently determined by the V (eq) of the Li-NTL solution as a consequence of the dissociation equilibrium of the ion pair of the solvated Li+ ion and [NTL](center dot-) radical ions.

Cell therapy using human-stem-cell-derived pancreatic beta cells (hSC-βs) is a potential treatment method for type 1 diabetes mellitus (T1D). For therapeutic safety, hSC-βs need encapsulation in grafts that are scalable and retrievable. In this study, we developed a lotus-root-shaped cell-encapsulated construct (LENCON) as a graft that can be retrieved after long-term hSC-β transplantation. This graft had six multicores encapsulating hSC-βs located within 1 mm from the edge. It controlled the recipient blood glucose levels for a long-term, following transplantation in immunodeficient diabetic mice. LENCON xenotransplanted into immunocompetent mice exhibited retrievability and maintained the functionality of hSC-βs for over 1 year after transplantation. We believe that LENCON can contribute to the treatment of T1D through long-term transplantation of hSC-βs and in many other forms of cell therapy.

Transplantation technologies of pancreatic islets as well as stem cell-derived pancreatic beta cells encapsulated in hydrogel for the induction of immunoprotection could advance to treat type 1 diabetes mellitus, if the hydrogel transplants acquire retrievability through mitigating foreign body reactions after transplantation. Here, we demonstrate that the diameter of the fiber-shaped hydrogel transplants determines both in vivo cellular deposition onto themselves and their retrievability. Specifically, we found that the in vivo cellular deposition is significantly mitigated when the diameter is 1.0 mm and larger, and that 1.0 mm-thick xenoislet-laden fiber-shaped hydrogel transplants can be retrieved after being placed in the intraperitoneal cavities of immunocompetent diabetic mice for more than 100 days, during which period the hydrogel transplants can normalize the blood glucose concentrations of the mice. These findings could provide an innovative concept of a transplant that would promote the clinical application of stem cell-derived functional cells through improving their in vivo efficacy and safety.

Human iPSC-derived hepatocytes hold great promise as a cell source for cell therapy and drug screening. However, the culture method for highly-quantified hepatocytes has not yet been established. Herein, we have developed an encapsulation and 3D cultivation method for iPSC-hepatocytes in core-shell hydrogel microfibers (a.k.a. cell fiber). In the fiber-shaped 3D microenvironment consisting of abundant extracellular matrix (ECM), the iPSC-hepatocytes exhibited many hepatic characteristics, including the albumin secretion, and the expression of the hepatic marker genes (ALB,HNF4 alpha,ASGPR1,CYP2C19, andCYP3A4). Furthermore, we found that the fibers were mechanically stable and can be applicable to hepatocyte transplantation. Three days after transplantation of the microfibers into the abdominal cavity of immunodeficient mice, human albumin was detected in the peripheral blood of the transplanted mice. These results indicate that the iPSC-hepatocyte fibers are promising either asin vitromodels for drug screening or as implantation grafts to treat liver failure.

This paper describes probiotic microfibers for oral delivery to reestablished healthy microbiome (Fig. 1). Recent discoveries in biology and microbiology have gained much importance of the gastrointestinal microbiome in regulating human health and disease. In this study, we fabricate core-shell probiotic microfiber for oral delivery. This microfiber can be encapsulated live bacteria into the center of the microfiber covered with hydrogels, and thus the bacteria can grow and form the biofilm-like structure. As a demonstration, the bacteria in the probiotic fiber survived after acid stimulation and oral dosing into the rat.

In this paper, we demonstrate that hepatocytes derived from human pluripotent stem cells (iPSCs) could promote their cell characteristics in cell-laden core-shell microfiber. The iPSC-hepatocytes are expected as cell source for regenerative medicine and chemical screening in drug development, but the cells are known to lose their properties in conventional two-dimensional (2D) culture immediately. Here we show that the iPSC-hepatocytes could be encapsulated into extracellular-matrix (ECM)-rich three-dimensional (3D) microenvironment by using microfluidic device system (named as "cell fiber technique"), and highly expressed hepatocyte-associated proteins and genes in the fibers. Furthermore, the fibers could be useful as functional grafts. These results indicate that the cell fiber can be applicable to in vitro construction of 3D hepatic tissue owing to spatial regulation of culture environment.

We developed a continuous glucose monitoring (CGM) system for the monitoring of 3D tissue in vitro using a perfusable device. The system consists of perfusable cell culture unit and CGM unit that has a fluorescent-based glucose responsive hydrogel sensor, LED and a photodiode on a chip; the unit is placed under a fluidic channel. This system enables in situ observation of the glucose concentration in culture medium to control the environment of the culture systems. We confirmed that the fluorescence intensity increased depending on the glucose concentration; ranging from 0 mg/dL to L000 mg/dL. We successfully cultured the 3D tissue in the perfusable unit and monitored the glucose concentration of medium for 24 hours.

This paper describes a multicorc-shcll hydrogel fiber encapsulating multi type cell with lotus root structure (Fig. 1). Microfluidic techniques are able to encapsulate the cells in 3D configuration such as beads and fibcrs. Especially, an excellent method was reported that the core-shell cell microfibers reconstitute intrinsic morphologies and functions of living tissues in vitro [1]. Among various tissucs and organs that have been targeted for in vitro reconstruction using cell microliber application, various types of cells, such as pancreatic 3 cells [2], adipocytes [3], neural cells [4] and stem cells [5], can bc cultured in a 3D tubular hvdrogel microenvironment. However, cell distribution in vivo is extremely complex, and spatially anisotropic or heterogeneous tissues are N\ idc sprc a d. We here took an approach to fabricate multicore-shell fibcrs with controlled heterogeneous cell-laden structures for construction of macro-size tissue. The fibcrs mimicked the structure of lotus root and the encapsulated cells in the fiber separately cultured as multi cell type co-culture system.

In this paper, we found that hepatocytes derived from human pluripotent stem cells (iPSCs) could promote their cell characteristics in cell-laden core-shell microfiber. The iPSC-hepatocytes are expected as cell source for cell therapy and drug testing, but the cells lose their properties in conventional two-dimensional (2D) culture. Here we demonstrate that the iPSC-hepatocytes could be encapsulated into extracellular-matrix (ECM)-rich three-dimensional (3D) microenvironment by using microfluidic device, and highly expressed hepatocyte-specific genes. This indicates that the cell fiber technique can be useful for generation of highly functional hepatic tissue in vitro owing to spatial regulation of culture environment.

This paper proposes a microfiber-shaped anode for enhancing performance of miniaturized microbial fuel cell (μMFC) system. MFC has a potential to directly generate electricity from organic substances. However, it remains challenging to obtain high current and power density for μ.MFC, due to small active area and electrochemical catalytic activity of their anodes. In this study, we developed fiber-shaped anode electrode utilizing core-shell fiber technique. Nutrients can easily penetrate into core because the diameter of the anode is micro-size. In addition, core of exoelectrogens is surrounded by shell of carbon nanotube (CNT) and hydrogel. Therefore, exoelectrogens can attach on CNT directly and form biofilm. Then, this performance of biocatalytic activity was confirmed by cyclic voltammetry (CV). We believe that our microfiber-shaped electrode will allow increasing the power density of μMFC system.

This paper describes a 3D perfusable device for the purification of pancreatic islets and the assessment of dynamic insulin secretion of purified islets. (Fig. 1) Islet transplantation is an innovative treatment that have the advantages such as patient safety, simplicity and minimally invasiveness, compared to pancreatic transplantation. However, it is difficult to collect a sufficient number of islets for transplantation and to evaluate sufficient quality check of isolated islet. In this study, we present fabricated a perfusable device for functional assays of the small number of islets by using 3D fluidic systems. In the system, the pancreatic islets were purified to high purity pancreatic β-cells and remove exocrine pancreas cells. Furthermore, purified islets were stimulated with different glucose concentration buffer and stimulated islets secreted the insulin characterizing their functionality.

A novel and simple method for constructing cell sheets is described in which culture surfaces were modified with RGD peptide-coupled alginate hydrogels using an electrodeposition method. Cells were cultured on the hydrogels to form a contiguous cell sheet-like structure. Then, the hydrogels were dissolved by addition of an EDTA solution and the cell sheets rapidly detached from the surface. This is the first report of the fabrication of cell sheets on RGD peptide-coupled alginate hydrogels using an electrodeposition method. We believe the technique is useful for cell sheet engineering.

Microencapsulation of cells is a promising technique in biomedical applications such as cell therapy. Recently, cell-laden hydrogel microfibers have been proposed as another shape microcapsule instead of microbeads; however, these are brittle with little stretching capability. This paper describes a cell-laden hydrogel microfiber that showed enhanced mechanical properties and handleability by using a double network (DN) hydrogel consisting of alginate and polyacrylamide. The DN hydrogel microfiber supported approximately 6 fold higher strain and exhibited 10-fold higher tensile strength than the conventional alginate form. The DN hydrogel gelation microfiber could also encapsulate pancreatic beta cells while maintaining cell viability and function. The in vivo functionality of the DN hydrogel microfiber was demonstrated by transplanting 3D assemblies of the microfibers into the intraperitoneal or subcutaneous space of diabetic mice, which successfully decreased their blood glucose levels. Thus, cell-laden DN hydrogel microfibers may represent a promising material for various biomedical applications.

This paper describes a core-shell hydrogel microfiber consisted of alginate and tetra-arm poly(ethylene glycol) (tetra-PEG) encapsulating rat insulinoma cells using a microfluidic process with a coaxial device and rapid gelation induced by diffusion of two different cross-linkers of barium ion and dithiothreitol (DTT) in the laminar flow (Fig. 1). Microfluidic techniques are able to encapsulate the cells in 3D configuration such as beads and fibers. For the medical treatment, an excellent method was reported that the retrievable graft of rat primary islet cell fiber for the treatment of diabetic mice [1]. However, the microfiber constructed of alginate hydrogel becomes weak of the strength, this issue is important for long-term transplantation. We here took an approach to improve the material, and developed composite hydrogel microfibers encapsulating cells with high mechanical strength. Using the double-network hydrogel consisted of barium-alginate and tetra-PEG as microfibers shell, the microfibers were able to encapsulate cells and maintained their shape without broken for 14 days intraperitoneal transplantation.

Here we propose a novel electrochemical lithography methodology for fabricating calcium-alginate hydrogels having controlled shapes. We separated the chambers for Ca2+ production and gel formation with alginate with a semipermeable membrane. Ca2+ formed in the production chamber permeated through the membrane to fabricate a gel structure on the membrane in the gel formation chamber. When the calcium-alginate hydrogels were modified with collagen, HepG2 cells proliferated on the hydrogels. These results show that electrochemical hydrogel lithography is useful for cell culture.

© 15CBMS-0001. This conference proceeding describes a wearable biosensor based on enzyme-modified carbon nanotube (CNT) microfibers for simple colorimetric detection of any target. Conventional biosensors based on enzyme-modified nanomaterials such as CNT mainly used for electrochemical detection [1-3]. The electrochemical system offers with high sensitivity, however the system requires external equipment for electrical measurements. Color detection enables simple and quick analysis without external equipment, but it is difficult to identify colors of enzyme-modified CNT biosensor with naked eyes. To solve this problem, it is expected to develop simple and smart wearable biosensors for anyone use. In this study, we demonstrate a biosensor based on an enzyme-modified CNT in a hydrogel microfiber. This fiber can be patterned on a commercially available "tattoo seal", and thus it can be easily attach to human skin. The color of the microfiber was changed to blue-violet by reacting with various targets including glucose, lactate, and alcohol; this reaction can be observed with naked eyes. We believe that this wearable biosensor has a possibility to be applied to simple/continuous health monitoring.

© 14CBMS. This conference proceeding describes a highly stretchable cell-laden hydrogel microfiber using doublenetwork hydrogels. Previously, a useful method involving the implantation of rat primary islet cell fibers for the treatment of diabetic mice was reported [1]. However, normal microfiber constructed from alginate hydrogel shell is too weak to be handled by hand manipulation. We here develop an approach to improve the shell material and developed highly stretchable cell-laden hydrogel microfibers. Using the doublenetwork hydrogel consisted of alginate-calcium and polyacrylamide as the microfiber's shell, the microfibers showed high stretchability with about 6 times higher than that of the existing hydrogel microfibers. The highly stretchable cell fibers would be useful for the application in robust suture-type tissue grafts and the double-network hydrogel shell serve a function of immunity isolating membrane.

© 14CBMS. In the present study, we present a method of eletrodeposittion of hydrogels to fabricate cell sheets. In the method, calcium-alginate hydrogels are electrodeposited by water electrolysis. 3T3 cells were cultured on the hydrogels to form cell sheets, and the cell sheets were collected by dissolving the hydrogels. Intact cell sheets can be collected, indicating that the method is useful to create cell sheets.

A local redox cycling-based electrochemical (LRC-EC) chip device was used to investigate the relationship between cardiomyocyte differentiation from embryonic stem (ES) cells and alkaline phosphatase (ALP) activity. In the LRC-EC chip device, ring-type interdigitated array electrodes were incorporated at n x n measurement points with only 2n bonding pads for external connection. Microwells were also fabricated at each measurement point to trap cell aggregates. To differentiate ES cells into cardiomyocytes, ES cells were three-dimensionally cultured to form simple and cystic embryoid bodies (EBs). ALP activity of these EBs was then detected using the LRC-EC chip device. The electrochemical responses for ALP activity decreased concurrently with the differentiation of ES cells into cardiomyocytes, indicating that an LRC-EC chip device is useful for evaluating cell differentiation. (c) The Electrochemical Society of Japan, All rights reserved.

In this study, we developed a novel method for fabricating microwell arrays constructed from alginate gels, and the alginate gel microwells were used for three-dimensional (3D) cell culture. The alginate gel microwells were fabricated on a patterned ITO electrode using alginate gel electrodeposition. Embryonic stem (ES) cells or hepatocellular carcinoma cells (HepG2) were cultured in the alginate gel microwells containing 3T3 cells. During the culture, embryoid bodies (EBs) or HepG2 spheroids were successfully fabricated in the alginate gel microwells. The oxygen consumption of the EBs indicated that they were successfully cultured. Liver-specific gene expressions of the HepG2 spheroids apparently increased by performing 3D co-culture in the microwell arrays with 3T3 cells. These results show that the alginate gel microwells are a useful 3D culture system.

In this study, tubular hydrogel structures were constructed via electrodeposition using alginate gels. Electrolysis of water in alginate solutions with calcium carbonate particles induced gel aggregation around Pt wire electrodes, forming tubular alginate gel structures. The simple method is a promising approach for construction of multi-layer tubular hydrogel structures for tissue engineering. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

In this study, a useful method was developed to fabricate array patterns of microparticles not on electrode surfaces, but on arbitrary surfaces, using negative-dielectrophoresis (n-DEP). First, electrodes were designed and electric field simulations were performed to manipulate microparticles toward target areas. Based on the simulation results, multilayered array and grid (MLAG) electrodes, consisting of array electrodes surrounded by insulated regions and a grid electrode, were fabricated for the formation of localized, non-uniform electric fields. The MLAG electrode was mounted to a target substrate in a face-to-face configuration with a spacer. When an AC voltage (4.60 V-rms and 1 MHz) was applied to the MLAG electrode, array patterns of 6 and 20 mu m diameter microparticles were rapidly fabricated on the target substrate with ease. The results suggest that MLAG electrodes can be widely applied for the fabrication of biochips including cell arrays. Biotechnol. Bioeng. 2009;104: 709-718. (C) 2009 Wiley Periodicals, Inc.