The mesothelium is a membrane that forms the lining of several body cavities. The mesothelium forms a monolayer of flattened mesothelial cells resting on a thin basement membrane. During continuous ambulatory peritoneal dialysis, the peritoneum is constantly exposed to bioincompatible dialysis fluids that cause denudation of mesothelial cells, and ultimately tissue fibrosis. In the present study, we have manufactured the human mesothelial cell sheet with the characteristics of the native mesothelium, using our cell sheet-based tissue engineering approach. A decrease in culture temperature released cells (cell sheet) from the temperature responsive culture dish surfaces without the need for enzymatic or chelation treatments. The mesothelial cell sheet possessed the characteristics of normal mesothelial cells, and showed abundant microvilli. The cell sheet was transplanted on normal perietal peritoneum using nude mouse. The cell sheet was remained on perietal peritoneum at a day later after the transplantation. The mesothelial cell sheet can be applied to repair the damaged peritoneum.

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Regenerative medicine aims to regenerate damaged tissues and organs by replacing them with tissue substitutes grown and reconstructed in the laboratory. One of the potential methods for reconstructing well-organized tissues in vitro is the use of cellular building blocks, such as cell sheets and spheroids. The fabrication and manipulation of such building blocks rely on innovative strategies for noninvasive cleavage of cell-to-culture substrate connections while preserving cell-to-cell connections. Several examples toward this end including our electrochemical approach have been presented. In our approach, cells are cultured on self-assembled monolayers (SAMs) of alkanethiols or oligopeptides adsorbed on a gold surface. The SAM was then reductively desorbed from the gold substrate by the application of a negative electrical potential. This caused the cells to detach from the gold surface in a rapid and reliable manner while maintaining cell-cell connections. This technology can be used to fabricate cell sheets, spheroids, and vascular-like structures.

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Oxygen supply is a most important issue in liver tissue engineering for regenerative medicine and cell-based assays. Here, we described our latest results on the use of oxygen-permeable membrane for these applications. Such a membrane enables direct oxygen supply to cultured liver-derived cells from the surrounding gas phase. This resulted in a realization of aerobic respiration of the cells, leading to spontaneous organization to multilayered seudo-3D tissues. In addition, precise control of actual oxygen concentration at the cell level was shown to be very important in the growth and maturation of liver progenitor cells.

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Embryonic stem cell (ES cell) and induced pluripotent stem cell (iPS cell) have an almost unlimited capacity for self-renewal and potential to differentiate into multiple cell lineages. These cells have much attention as a cell source for a regenerative medicine technology. To realize the therapeutic potential of these stem cells, it is necessary to establish an effective differentiation method from undifferentiated stem cell to specific matured cell. In this study, we investigated the hepatic differentiation of mouse ES cells by the applying hollow fiber (HF)/organoid culture method, in which cultured cells form a cellular aggregate called an “organoid” in the lumen of the HF. ES cells showed high proliferative activity immediately after cell seeding. The expressions of liver-specific functions were detected after about 2 weeks of the hepatic differentiation induction in the HF/organoid culture. As an application of this differentiation technique to regenerative medicine, we designed a hollow fiber-type bioartificial liver device. Because of high proliferative activity of ES cells and high cell density, the maximum expression level of liver-specific functions per unit volume of the device were comparable to that seen in primary mouse hepatocyte culture. In conclusion, the HF/organoid culture method has promise as an effective tool for the differentiation of ES cells into hepatocytes.

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Pluripotent stem cells (ES / iPS cells) have the potential to differentiate into any of the germ layers, holding great promise not only for a model of early embryonic development but also for provision of robust cells for regenerative medicine and drug discovery. Three-dimensional spherical cell aggregates called embryoid bodies (EBs) have been widely used in in vitro differentiation protocols of pluripotent stem cells, from which even self-organized three- dimensional tissues and organs such as eye-cups and adenohypophysis have been obtained. Directed differentiation of pluripotent stem cells via EB formation is affected by the cell number, size and shape of EBs, requiring strict quality control of EBs. The hanging drop method, which is a conventional technique for EB formation, is suitable for forming well-controlled EBs but is inherently incapable of large-scale production. On the other hand, the static suspension culture method exhibits limited control over cell aggregation. Recently, non-cell-adhesive culture vessels have been developed as effective tools for EB formation. Culture vessels coated with phospholipid polymer (Lipidure®-Coat) perform excellent anti-adhesive property, enabling efficient production of well-controlled EBs by quite simple operation. The first objective of this review is to summarize the technological development of simplified methods for EB formation. The second objective is to describe the development and applications of Lipidure®-Coat.

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Biomembranes, typified by an ion channel such as a K⁺ channel, can recognize a specific ion, and can control the transport of recognized ions by themselves. Inspired by the functions of biomembranes, we have fabricated a molecular recognition ion gating membrane. The membrane was prepared by plasma graft copolymerization, which filled the pores of porous polyethylene film with a copolymer of NIPAM (N-isopropylacrylamide) and BCAm (benzo[18]crown-6-acrylamide). NIPAM is well known to have an LCST (lower critical solution temperature), at which its volume changes dramatically in water. The crown receptor of the BCAm traps a specific ion, and causes a shift in the LCST. Therefore, selectively responding to either K⁺ or Ba²⁺, the grafted copolymer swelled and shrank in the pores at a constant temperature between two LCSTs.
We have been studying the interesting functions of the membrane. The membrane changed its permeation flux in response to the ion signals. The solution flux in the absence of Ba²⁺ decreased by about two orders of magnitude over a solution flux containing Ba²⁺. Osmotic pressure through the membrane was changed in response to the ion signals. The membrane controlled the diffusion of model drugs in response to the ion signals. Due to its stability, the membrane also showed responding function in the mixture of ethanol and water. As typified by Hodgkin-Huxley equation, excitation of neuron synapses is an interesting phenomenon of biomembranes. Three ion channels work cooperatively and make dynamics of membrane potential. Theorell made the oscillator of the artificial glass membrane, hinted by neurons. Inspired by Theorell oscillator, we combined hydrodynamic pressure-driven permeation and osmotic permeation through the membrane. We finally found that the membrane showed autonomous oscillation in response to Ba²⁺ only.

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Novel carbon hollow fiber membranes were successfully fabricated using poly(phenylene oxide) (PPO) and its functionalized derivatives as a polymeric precursor. These carbon membranes had properties above the upper bound of conventional polymeric membranes reported by Robeson including PPO polymeric precursors. Metal containing carbon membranes derived from sulfonated PPO (SPPO) were prepared by ion exchange method and the effect of metal cations on the gas transport properties was studied. We found that the incorporation of transition metal ions such as Ag+ significantly enhanced gas permeabilities without affecting the ideal selectivities. We also found that a simple modification of the PPO precursor with a TMS group enhanced gas permeabilities of carbon membrane. It can be considered that the incorporation of silica-like species into the carbon membranes was formed and it increased the apparent free volume of the carbon matrix. The development of flexible carbon hollow fiber membranes was successfully achieved using SPPO as a precursor polymer. The superior flexibility combined with excellent gas separation performance were obtained when pyrolyzed at 600 °C. This result presents a promising possibility for the advancement of commercial application of carbon membranes. Selective membrane separation of saturated water vapor from olefin gas such as ethylene and propylene has been investigated using two types of membrane modules based on SPPO carbon hollow fibers.

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The response of zeta potential,ζ_obs, to membrane surface condition and surfactants in solution was investigated from the viewpoint of the interaction between surfactant and membrane surface. The ζ_obs of three polyvinylidene difluoride (PVDF) microfiltration membranes, which had non-ionic hydrophilic, hydrophobic, and positively charged pore surfaces, with nominal pore size of 0.22 μm were measured over wide range of the surfactants concentration. N-Dodecyltrimethyl ammonium chloride (DTAC) and Sodium Dodecyl Sulfate (SDS) were used as cationic and anionic surfactants, respectively. At the lower surfactant concentration (the conductivity below 20mS/m) the behaviors of ζ_obs mainly depended on the combination of membrane and surfactant used and they can be explained by the interaction between surfactant and pore surface. At the higher surfactant concentration the behaviors of ζ_obs mainly depended on type of surfactant rather than on membrane used. In the case of SDS the severe flux decline was observed at a certain concentration in all the membranes and the absolute value of ζ_obs decreased and approached to zero reflecting the flux decline.

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A new cosmetic series VIVCO was commercialized by utilizing the solid-in-oil (S/O^®) technique for L-ascorbic acid phosphate magnesium salt (vitamin C derivative) and hyaluronic acid. In the S/O^® technique, active ingredients are coated by hydrophobic surfactant molecules and the treatment changes their surface properties from hydrophilic to hydrophobic one. Therefore the coated active component can be dissolved in various types of oils and we can get homogeneous cosmetic oil. As a result, the cosmetic oil containing the surfactant-coating ingredient is effective for enhancing the permeation of active components into the deep region of the skin, because the outer surface of skin is hydrophobic. The S/O^® technique was also found to be useful for stabilizing the ingredients by covering with a surfactant layer.

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