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Insufficient reliability of current drug screening by cell-based assay has been one of the factors in the poor success rate in drug development. To improve the situation, we proposed a cell culture system using semi 3-D hydrogel microstructures as cell culture scaffolds. Because of its flexibility and permeability, the microstructure was expected to enhance the physiological function of cells. We developed a simple method of fabricating the unique hydrogel microstructures composed of hydroxypropyl cellulose through photolithography. Functionalization with poly(styrene-co-maleic anhydride) gained their cell adhesion, and it was demonstrated that several kinds of cells were incubated on the cell culture scaffolds.

The use of organ-on-a-chip (OOC) devices is a promising alternative to existing cell-based assays and animal testing in drug discovery. A rapid prototyping method with polydimethylsiloxane (PDMS) is widely used for developing OOC devices. However, because PDMS tends to absorb small hydrophobic molecules, the loss of test compounds in cell-based assays and increases in background fluorescence during observation often lead to biased results in cell-based assays. To address this issue, we have fabricated a glass-based OOC device and characterized the medium flow and molecular absorption properties in comparison with PDMS-based devices. Consequently, we revealed that the glass device generated a stable medium flow, restricted the absorption of small hydrophobic molecules, and showed enhanced cell adhesiveness. This glass device is expected to be applicable to precise cell-based assays to evaluate small hydrophobic molecules, for which PDMS devices cannot be applied because of their absorption of small hydrophobic molecules.

This study aims to investigate bacteriorhodopsin (bR) molecules reconstituted in lipid bilayers composed of di(nonafluorotetradecanoyl)-phosphatidylcholine (F4-DMPC), a partially fluorinated analogue of dimyristoyl-phosphatidylcholine (DMPC) to clarify the effects of partially fluorinated hydrophobic chains of lipids on protein's stability. Calorimetry measurements showed that the chain-melting transition of F4-DMPC/bR systems occurs at 3.5 °C, whereas visible circular dichroism (CD) and X-ray diffraction measurements showed that a two-dimensional (2D) hexagonal lattice formed by bR trimers in F4-DMPC bilayers remains intact even above 30 °C, similar to bR in a native purple membrane. Complete dissociation of the trimers into the monomers detected by visible CD almost coincides with the complete melting of 2D lattice observed by X-ray diffraction, in which both take place at around 65 °C (10 °C lower than that for bR in a native purple membrane). However, it is extremely high in comparison with the bR reconstituted in DMPC bilayers in which the dissociation of bR trimer in DMPC bilayers occurs near the chain-melting transition temperature of DMPC bilayers at approximately 18 °C. In order to explore the rationale behind the difference in stability, a further investigation of the detailed structural features of pure F4-DMPC bilayers was performed by analyzing the lamellar diffraction data using simple electron density models. The results suggested that the perfluoroalkyl groups do not exhibit any conformation change even if the chain-melting transition occurs, which is likely to contribute to the stability of the 2D hexagonal lattice formed by the bR trimers.

We report a sharp photoinduced aqueous dissolution of the copolymer through phase transition based on the photochemical reaction of o-nitrobenzaldehyde (NBA) and the principle of polymer effect. We synthesized the copolymers having poly( N-isopropylacrylamide) main chain and NBA side chain at 4, 7, and 10 mol % functionalizations and analyzed their photoresponsive characteristics. Light with 365 nm wavelength converted NBA groups at copolymer side chains to carboxylic acid efficiently at the rate of 7.3 cm2/J, and in the case of 10 mol % functionalization, the irradiation dosage no more than 56 mJ/cm2 induced sharp aqueous dissolution of the copolymer thin layer in pH 7.4 at 25 °C. As example applications, we demonstrated on-demand release of polyethylene beads and fluorescent-labeled albumins, which had been immobilized on a substrate surface via the copolymers, by the precisely controlled light irradiation using a microprojection system. Also, we examined application of the copolymers to the selective recovery of living cells from culture substrate under microscopic observation. As a result, mild light irradiation at room temperature triggered immediate detachment of the cultured adherent cells only in the irradiated areas without critical influence on their viability.

Photoresponsive actuators composed of a hydrogel functionalized with spiropyran are described. The hydrogel exhibits drastic shrinking in rapid response to blue light irradiation in acidic aqueous systems, and is examined for the application of several photoresponsive actuator systems. Rod-like hydrogel bends drastically after 1 s light irradiation, and microrelief is formed instantly on the hydrogel sheet by the micropatterned light irradiation. Based on these characteristics of the hydrogel, a photo-controllable microfluidic system is constructed with a hydrogel sheet, and the microchannels with arbitrary width, height and pathway are formed instantly by the micropatterned light irradiation. Also independent and parallel control of microvalve array by local light irradiation is demonstrated for a similar microfluidic system combined with fixed microchannel.

To examine the applicability of fluorinated membrane-forming phospholipids to reconstitution matrices for functional membrane proteins, the membrane properties of a synthetic ether-type phosphatidylcholine (PC) bearing partially fluorinated C18-monoacetylenic (9-octadecynyl) chains, DF8CCH8PC, were compared with those of its non-fluorinated counterpart, DH8CCH8PC. Light-harvesting complex 2 (LH2) and the light-harvesting 1‒reaction center core complex (LH1-RC) isolated from purple photosynthetic bacteria were employed as probe membrane proteins to evaluate the extent to which their reconstitution into DF8CCH8PC membranes could proceed. DF8CCH8PC formed more expanded and more stable fluid monolayers than DH8CCH8PC at the air-water interface at 25 °C; the former PC molecule occupied an area of ca. 0.70 nm2 at a collapse pressure, πc, of 52 mN/m, while the latter occupied an area of ca. 0.55 nm2 at a πc of 45 mN/m. In contrast, the molecular motion detected using fluorescent probes was much more restricted in DF8CCH8PC bilayers than in DH8CCH8PC ones. Although the reconstitution efficiencies of both LH2 and LH1-RC into DF8CCH8PC bilayers were lower than those into DH8CCH8PC bilayers, the membrane proteins incorporated into DF8CCH8PC bilayers showed increased thermostability. The increased thermostability of these proteins in fluorinated PC membranes might be due to the restricted molecular motion in the hydrophobic chains. The results of this study suggest that partially fluorinated PCs can be useful materials for the construction of lipid‒functional membrane protein assemblies including large membrane protein complexes, such as LH1-RC, for biotechnological applications.

Interfacial properties and membrane protein solubilization activity of a series of partially fluorinated octyl-phosphocholine (PC) surfactants were investigated from the viewpoint of the fluorination degree of the hydrophobic chain. The critical micelle concentration (CMC), surface tension lowering activity, molecular occupied area at the CMC and free energy changes of micellization as well as adsorption to the air-water interface for each PC surfactant were estimated from surface tension measurements at 25 °C. The PCs with higher degree of fluorination exhibited low CMC and high surface activity, while the single trifluoromethyl group at the end of the chain appeared to enhance the hydrophilicity of the surfactant molecule. Under conditions where conventional short-chain surfactants, n-octyl-β-D-glucoside, Triton X-100 and dioctanoylphosphatidylcholine significantly solubilize purple membranes (PM), none of the fluorinated-PCs solubilized PM. This suggests that fluorinated-PCs are low-invasive enough to maintain the structure of lipids/protein assemblies like PM.

Photolithographic fabrication of unique microstructures composed of flexible hydrogel sheets is proposed and demonstrated by using photo-acid-generating poly(methyl methacrylate). Crosslinking of a hydroxyl-rich polymer and lifting off of the crosslinked polymer layer from the substrate are controlled respectively in an area-selective manner upon micropatterned light irradiation, and various pocket-like microstructures are fabricated resultantly.

The photo- and thermoresponse of poly(N-isopropylacrylamide) (pNIPAAm) functionalized with spiropyran chromophore is examined with respect to the influence of molecular recognition by cyclodextrin (CD). Characterization in aqueous solutions of spiropyran-functionalized poly(N,N-dimethylacrylamide) under coexistence of α-, β-, or γ-CD reveals that β-CD selectively includes the ring-closing isomer of the chromophore, which is dominant under light irradiation, while no inclusion is observed for the protonated ring-opening isomer, which is dominant in the dark before irradiation. As a result, it is shown that the selective inclusion of the chromophore at a polymer side chain is switched by light irradiation. Further, drastic photoresponsive dehydration of spiropyran-functionalized pNIPAAm is inhibited only by β-CD out of three examined CDs, demonstrating that the molecular recognition regulates the dehydration of the whole polymer triggered by the photoswitching of the chromophore introduced at only 1 mol% functionalization.

The aggregation behavior of short-chained archaeal phospholipid analogs (DCn+mPCs, (n, m) = (4, 1) and (8, 2), n: number of carbon atoms in the single main chain; m: number of methyl branches in one chain. PC denotes phosphatidylcholine) in water was investigated as a function of the hydrophobic chain length and the glycerol-linkage type (ester or ether bond), using fluorescent probe method, polarized light microscopy and XRD. DC8+2PCs yielded myelin-figures in their dilute solutions at 25 °C, indicating that they tend to form vesicular structures rather than micelles. In contrast, DC4+1PCs lacked to form myelin-figures in their dilute solutions, being similar to short straight-chained PC surfactants, e.g., dioctanoyl-PC. The critical micelle concentrations of DCn+mPCs could be well interpreted on the basis of the total water contact surface area of the lipid hydrophobic chains, while the glycerol-linkage type seemed to have only a minor effect on the aggregation behavior of lipids.

A closed-ring isomer of a diarylethene having a sulfone group works as the reagent for SO2 gas generation with thermal stability even at 70 °C, and it rapidly reverts to the open-ring isomer and generates the SO2 gas to induce cell death upon UV irradiation.

The mixing behavior of hydrated dimyristoylphosphatidylcholine and its partially fluorinated analogue, di(nonafluorotetradecanoyl)phosphatidylcholine, where all hydrogens of the terminal n -butyl segments in two hydrophobic chains are replaced by fluorines, has been investigated by calorimetry and X-ray diffraction. The analysis of the calorimetric data by using the regular solution theory has revealed that the phase diagram of the mixtures is a eutectic type with a miscibility gap. The non-ideality parameters of the gel and liquid-crystalline phases are estimated to be 5.7 ± 0.1 and 4.2 ± 0.1 kJ/mol, respectively. These large values indicate that the mixing is significantly non-ideal. X-ray diffraction has shown that a phase separation occurs in the miscibility gap.

Fluorinated lipids and surfactants are attractive biomimetic materials for the extraction and reorganization of membrane proteins because of the biological inertness of fluorocarbons. We investigated the fundamental physical properties of a partially fluorinated phospholipid (F4-DMPC), such as phase transition, area thermal expansion, and lateral lipid diffusion, to evaluate the intermolecular interaction of F4-DMPC in the hydrophobic region quantitatively on the basis of free-volume theory. Fluorescence microscope observation of the supported lipid bilayer (SLB) of F4-DMPC showed that the phase transition between the liquid crystalline and gel phases occurred at 5 °C and that the area thermal expansion coefficient was independent of the temperature near the phase transition temperature. We performed a single particle tracking of the F4-DMPC-SLB on a SiO2/Si substrate, to measure the diffusion coefficient and its temperature dependence. The apparent activation energy (E′a) of lateral lipid diffusion, whic...

Ordinary fatty acids such as oleic, elaidic and stearic acids exist as their hydrogen-bonded dimers in their liquids and in non-polar solvents. Infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy have revealed that semi-fluorinated (SF) acids containing a perfluorooctyl group (C₈F₁₇) as a terminal segment exist also as hydrogen-bonded dimers, which are the units of inter- and intramolecular movements in their liquids and CCl₄. The dynamic molecular properties, such as self-diffusion coefficients and intramolecular movements of SF-oleic, SF-elaidic, and SF-stearic acids were compared with those of corresponding ordinary fatty acids (H-acids). From the high equilibrium spreading pressures (ESPs) for SF-acids compared with those for their corresponding H-acids, it was expected that the inter-acyl chain interaction is weaker for the SF-acid than for the H-acid: the SF-fatty acids should have higher molecular mobility than the corresponding ordinary H-acids in the liquid state. However, the self-diffusion coefficients obtained for SF-acids were smaller than those for the corresponding H-acids; the apparent activation energies for the self-diffusion process (translational movement) of SF-acids were larger than those for the corresponding H-acids. Namely, the motion of SF-acid molecules in a liquid phase is rather restricted compared with H-acid in spite of lower inter-acyl chain interaction of SF-acid. This unexpected result suggests that the molecular motion of SF-acid in a liquid phase is not directly governed by inter-acyl interaction, but may be interpreted as a reptation movement of an acid molecule, which is related to intramolecular movement. In fact, low intramolecular movements for SF-acid were confirmed by ¹³C-NMR T₁ measurements.

This article reviews our approaches for designing fluorinated lipid liquid–crystal membranes suitable for biomaterials such as membrane protein reconstitution matrices. We proposed several partially fluorinated fatty acids as well as phospholipids bearing long (C18) hydrophobic chains comparable to biomembrane lipids. The fatty acids and/or phospholipids containing perfluoroalkyl group was easily synthesized in good yields. Judging from the collapse (equilibrium spreading) pressures of the proposed fatty aids and/or phospholipids at the air–water interface, the partial fluorination of hydrophobic chains was enough to improve their monolayer stability. The fluorinated phospholipid with C–C triple bonds formed flexible structures such as stable vesicles in water at a room temperature. The partially fluorinated phospholipids are applicable to protein reconstitution matrices, e.g., reconstitution of photosynthetic membrane proteins.

The control of protein adsorption on microchannel surfaces is important for biosensors. In this study, we demonstrated protein adsorption method that is controlled through temperature change, i.e., thermoresponsive protein adsorption, on polydimethylsiloxane (PDMS) microchannel surfaces using a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm). To provide general protein adsorption control method, we adopted biotin-streptavidin chemistry and synthesized streptavidin covalently modified with PNIPAAm (PNIPAAm-StAv). Modification of streptavidin, a hydrophilic protein, with PNIPAAm induced successful thermoresponsive adsorption on a PDMS microchannel surfaces: PNIPAAm-StAv adsorbed at 37 degrees C and desorbed at 10 degrees C on the surfaces. We also demonstrated the thermoresponsive adsorption of biotinylated immunoglobulin G (IgG-b) using PNIPAAm-StAv. Conjugation of IgG-b with PNIPAAm-StAv induced successful thermoresponsive IgG-b adsorption on PDMS. Modification of PDMS surfaces with PNIPAAm reduced physical adsorption of the partially hydrophobic IgG-b on the surface and contributed to the high-contrast thermoresponsive adsorption of IgG-b: less than 1% of the IgG-b adsorbed at 37 degrees C was detected after the PNIPAAm-PDMS surface was washed at 10 degrees C. The controllable adsorption of this system is expected to be applied to the regeneration of biosensor chips and to on-chip protein manipulation.

In this study, we applied photo-induced graft polymerization to micropatterned surface modification of polydimethylsiloxane (PDMS) with poly(ethylene glycol). Two types of monomers, polyethylene glycol monoacrylate (PEGMA) and polyethylene glycol diacrylate (PEGDA), were tested for surface modification of PDMS. Changes in the surface hydrophilicity and surface element composition were characterized by contact angle measurement and electron spectroscopy for chemical analysis. The PEGMA-grafted PDMS surfaces gradually lost their hydrophilicity within two weeks. In contrast, the PEGDA-grafted PDMS surface maintained stable hydrophilic characteristics for more than two months. Micropatterned protein adsorption and micropatterned cell adhesion were successfully demonstrated using PEGDA-micropatterned PDMS surfaces, which were prepared by photo-induced graft polymerization using photomasks. The PEGDA-grafted PDMS exhibited useful characteristics for microfluidic devices (e.g. hydrophilicity, low protein adsorption, and low cell attachment). The technique presented in this study will be useful for surface modification of various research tools and devices.

We investigated hydration and swelling behavior of a solid state photoresponsive copolymer in water by using a quartz crystal microbalance technique with dissipation measurement (QCM-D technique). On the gold film electrode of a quartz resonator, we deposited a thin layer of a pNSp–NIPAAm, which is a poly( N -isopropylacrylamide) (pNIPAAm) polymer partially modified with a photochromic chromophore, 6-nitrospiropyran (NSp). Using QCM-D measurements, we found that at a temperature of 19 °C both water adsorption and changes in the viscoelasticity of the solid pNSp–NIPAAm layer were induced when pNSp–NIPAAm was irradiated by 365 nm ultraviolet light, which triggers the photoisomerization of the NSp chromophore and makes the structure of the chromophore hydrophilic. At temperatures between 25 and 35 °C, this photo-induced hydration was not observed. These observations suggest that the photoisomerization of the NSp chromophores triggered the photo-induced hydration only when pNIPAAm component is sufficiently hydrophilic, at a temperature of 19 °C.

Highly fluorinated single-chained and/or double-chained phospholipids containing the perfluorooctyl group as the terminal segment of hydrophobic chains and a phosphocholine moiety as the hydrophilic headgroup were synthesized in order to investigate the effect of fluorinated segments on the stability of phospholipid monolayers formed at the air–water interface. Judging from the equilibrium spreading pressures (πes) of their monolayers at the air–water interface, all of the fluorinated phospholipids formed more stable monolayers than the corresponding non-fluorinated counterparts. In addition, the fluorinated double-chained phosphatidylcholine containing C–C triple bond (monoyne group) formed stable and fluid vesicle membranes in water, although the single-chained phospholipids did not form vesicle membranes but micellar solutions under the present conditions.

This paper describes the generation of “click-crosslinkable“ and “photodegaradable“ gelatin hydrogels from the reaction between dibenzocycloctyl-terminated photoclevable tetra-arm polyethylene glycol and azide-modified gelatin. The hydrogels were formed in 30 min through the click-crosslinking reaction. The micropatterned features in the hydrogels were created by micropatterned light irradiation; the minimum resolution of micropatterning was 10-μm widths for line patterns and 20-μm diameters for circle patterns. Cells were successfully encapsulated in the hydrogels without any loss of viability across a wide concentration range of crosslinker. In contrast, an activated-ester-type photocleavable crosslinker, which we previously used to prepare photodegradable gelatin hydrogels, induced a decrease in cell viability at crosslinker concentrations greater than 1.8 mM. We also observed morphology alteration and better growth of cancer cells in the click-crosslinked photodegradable gelatin hydrogels that included matrigel than in the absence of matrigel. We also demonstrated micropatterning of the hydrogels encapsulating cells and optical cell separation. Both of the cells that remained in the non-irradiated area and the cells collected from the irradiated area maintained their viability.

The photo-generated closed-ring isomer of bis(5-methyl-2-phenylthiazoyl)perfluorocyclopentene shows cytotoxicity to Madin–Darby canine kidney (MDCK) cells through a caspase cascade and induces apoptosis of cells.

Living cells in human body exhibit their function in microstructured three dimensional (3D) environments composed of soluble factors, extracellular matrix (ECM), and neighboring cells. Fabricating biological 3D in vitro models of tissue and organ is a critical step for developing new strategies for cell based assay in drug discovery. To date, a number of research groups have developed micropatterning of hydrogels using photocurable and photodegradable hydrogels. This chapter reviews recent development on the techniques for microscale 3D fabrication of biomaterials by means of photolithography. Also, this chapter introduces an activated-ester-type photocleavable crosslinker, which we recently synthesized to generate photodegradable hydrogels using biocompatible materials such as polyethylene glycol and gelatin. This new type of crosslinker enabled convenient preparation of photodegradable hydrogel by two component mixing reaction. The hydrogels were degraded by micropatterned light irradiation, local light irradiation, and two-photon excitation. This simple and convenient approach to prepare and fabricate photodegradable hydrogels is creating new opportunity for novel cell manipulation and 3D tissue engineering techniques.

The facilitated transport mechanism of cerium(III) ions through polymer inclusion membrane (PIM) consisting of cellulose triacetate (CTA) as a polymer matrix, 2-nitrophenyl n -octyl ether (NPOE) as a solvent and N , N , N ′, N ′-tetraoctyl-3-oxapentanediamide (TODGA) as a carrier was studied. In order to evaluate the mass transport phenomena in the PIM, a mathematical model was derived from the Fick's first law and the equations for the extraction and the material balance. Methods to determine the values of the transport parameters such as the diffusion coefficient are described, and the dependency of the flux on the experimental condition was calculated. The model is very useful as a design tool to analyze and optimize the concentration process of low level radioactive wastewater using the PIM.

A photoresponsive hydrogel was prepared by radical copolymerization of N-isopropylacrylamide, a vinyl monomer having a spirobenzopyran residue and cross-linker. By the observation of photoresponsive shrinking and the conductance change, it was confirmed that the hydrogel in an acidic condition exhibited drastic and rapid volume shrinkage and proton dissociation when it was irradiated with blue light. Further, to examine its application to the mass transfer control, we prepared a photo- and thermoresponsive gate membrane by introducing this photoresponsive hydrogel to the surface of a porous membrane. As the first demonstration of the photocontrol of membrane permeation for liquid, it was observed that its permeability for 1 mM HCl aqueous solution increased by 2 times in response to the blue light irradiation, and this photoresponse of the permeability was confirmed to be repeatable.

Using a functional polymer containing nitrospirobenzopyran residues and poly(ethylene glycol), we developed a photoresponsive cell culture surface (PRCS), at which the cell adhesion can be enhanced locally by UV light irradiation (350–400 nm, 35 mW/cm2, 5 min) even on the region where living cells exist. Cell adhesion was evaluated by observing BALB/3T3 fibroblasts, which had been seeded on PRCS and then irradiated with UV light regionally, after 12 h incubation and subsequent washing to remove scarcely adhering cells. As a result, it was confirmed that the number of remaining cells in irradiated region was at most 2.5 times greater than that in nonirradiated regions, suggesting the implementation of a novel scheme to manipulate living cells individually by light irradiation in a parallel and simultaneous manner. Influence of the composition of the polymer material consisting the substrate surface was also investigated systematically. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 495–499, 2006

In this article, a new method to construct composites of hydroxyapatite (HAP) and polymer material is introduced. A previously developed method for mineralization of CaCO3 on a polymer substrate was applied to HAP. A solution that contained Ca2+, PO43–, and OH- ions was supersaturated with polyacrylic acid (PAA) that, at the same time, formed a polymer complex with the substrate, a polyvinyl alcohol (PVA) film, at the substrate surface. In this thin surface layer, nucleation of HAP took place. Subsequently, the disklike domains of HAP that were generated spread until they covered the PVA film surface. By regulating the pH of the supersaturated solution at around 7.4, the domain size decreased and the quantity of deposited material increased. Approximately 20 mg of HAP coating was obtained on a PVA film of 1 cm radius when the film was soaked in single 200 mL batch of the supersaturated solution for 21 days. The junction between HAP layer and PVA substrate film was found to be very firm. When a crosslinked PVA/PAA was used as the substrate, the film swelled in the supersaturated solution to form a hydrogel. Then mineralization took place within the gel, and a transparent monolithic composite of HAP and the polymer network was obtained. In 13 days, the weight increase was 29 mg, which corresponded to a 71 wt % HAP mineralization ratio of the composite. By changing crosslinking degree and HAP mineralization ratio, the flexibility of composite will be controlled in a wide range. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 1465–1470, 2006

Hydroxyapatite (HAP) was mineralized in poly(vinyl alcohol) (PVA)/poly(acrylic acid) (PAA) complex hydrogel immersed in a salt solution containing PAA. The transparent HAP/polymer composite swelled in water depending on the HAP content; high HAP content gave small swelling and vice versa. The HAP content reached about 80 wt % at most. Observation of the cross section of the composite by energy-dispersive analysis of X-ray (EDAX) revealed that the composite consisted of two phases, i.e., a hard HAP-rich phase and a soft polymer-rich phase. In the HAP-rich phase, the space inside the hydrogel was occupied by HAP, while HAP was not mineralized in the polymer-rich phase. The nucleation seemed to take place, at first, at the middle depth of the hydrogel where the HAP-rich phase was formed. The HAP-rich phase grew its size toward the surface of the hydrogel at the cost of the polymer-rich phase. The presence of phosphorus, P, in the polymer-rich phase indicated the adsorption of HPO(4)(2-) on the polymer chain of the hydrogel via hydrogen bonding, accompanied with Ca(2+) because of electrostatic constraints. This adsorption of ions in addition to Donnan distribution of ions leads to the formation of a hypercomplex that can be regarded as a precursor of the HAP-rich phase. The change of the hypercomplex into the HAP-rich phase is discontinuous and hence concluded as a phase transition. By comparison of our mineralization system with the biomineralization system of HAP and CaCO(3), the physicochemical mechanism of the mineralization process in the present system was found to be similar to that in biological systems. In this sense, we termed the present system an artificial biomineralization system.

This paper reports experimental results on pervaporation of benzene/cyclohexane mixtures and xylene isomer mixtures through a fixed-carrier membrane consisting of cellulose acetate (CA) as a base polymer and dinitrophenyl (DNP) group as a selective fixed-carrier. The result of the pervaporation of benzene/cyclohexane mixture using the unmodified CA membrane and the CA-DNP membrane showed that the DNP group was effective to increase the benzene selectivity. In the pervaporation of xylene isomer mixtures, the DNP group selectively facilitated the transport of xylene isomers through the membrane. The order of the preferentially permeating component was p -xylene > m -xylene > o -xylene.

A rapid and simple selection method of high-yield cells has been desired to establish highly productive cell lines for useful secondary metabolites. For this purpose, a new attempt was made to partition cultured plant cells in a poly(ethylene glycol)-dextran aqueous two-phase system (ATPS). The applicability of the ATPS in partitioning cultured strawberry cells (designated FAW) was investigated. The result of single-step partitioning in the ATPS supplemented with 0.4 mmol/kg lithium sulfate showed that FAW cells cultivated for 7 d under light-irradiation were separated into two cell populations with significantly different anthocyanin content. Additionally, the analysis technique of microscopic cell images showed that cells accumulating a high level of anthocyanin were partitioned completely into the bottom phase in a partitioning experiment of FAW cells cultivated for 10 d under light-irradiation in the ATPS supplemented with 1.8 mmol/kg potassium phosphate buffer. These results indicated that cell partitioning in ATPS increased the intracellular anthocyanin content and that the cultured strawberry cell population was heterogeneous in terms of cell surface properties. This is the first report of partitioning based on the heterogeneity of the cell surface properties correlated with the intracellular secondary metabolism in cultured plant cells. Our results also suggested that the ATPS was appropriate as a large-scale method for selecting useful cell lines among the cultured plant cells.

A fluorinated oleic acid 1 - Z containing a perfluorooctyl group and its analogues ( E -isomer 1 - E , alkyne type 2 and saturated type 3 ) were synthesized in good yields. In these syntheses, it was found that a key compound 5 could be converted to each of 1 - Z , 1 - E and 2 . Furthermore, equilibrium spreading pressures of their monolayers at the air–water interface were measured in order to demonstrate how the degree of unsaturation in the hydrophobic chain, the geometric isomerization, and the presence of F-atoms influence the monolayer stability. Irrespective of the structural alteration in the hydrophobic chains, the fluorinated fatty acids formed more stable monolayers with high spreading pressures as compared to their hydrocarbon counterparts.

Polymer inclusion membranes (PIMs) consisting of cellulose triacetate (CTA) as a polymer matrix, 2-nitrophenyl n -octyl ether (NPOE) as a solvent and octyl(phenyl)- N , N -diisobutylcarbamoylmethylphosphine oxide (CMPO) or N , N , N ′, N ′-tetraoctyl-3-oxapentanediamide (TODGA) as a carrier were developed, and the facilitated transport of cerium(III) ions using the PIMs was investigated experimentally. It was observed that the present PIMs were very effective to transport cerium(III) ions from the feed phase to the strip phase. The transport rate was strongly influenced by the experimental conditions such as the solvent/polymer ratio, the operating temperature, the membrane thickness, the carrier concentration and the feed concentration. The experimental results indicated that the transport was controlled by the diffusion of the ion-carrier complex in the membrane.

The dielectric environment surrounding poly(N-isopropylacrylamide) in aqueous solution was investigated by probing with spirobenzopyran covalently attached as a side chain to the polymer main chain. Inherent characteristics of the spirobenzopyran chromophore were analyzed, and the chromophore was confirmed to be suitable to probe the local polar condition around the polymer. Measurements for an aqueous polymer solution at various temperatures elucidated that the dielectric environment surrounding the polymer changed continuously even in the temperature range far below the lower critical solution temperature. This result suggested that the local and weak orientation of water molecules around the polymer diminished continuously in a preliminary stage of shifting to thermally induced phase separation. The dielectric environment surrounding thermoresponsive polymer in aqueous solution was investigated by probing with spirobenzopyran covalently attached as a side chain to the polymer main chain.

A novel functional copolymer was synthesized by modifying poly(N-isopropylacrylamide) with spirobenzopyran. The phase transition properties of the aqueous solution of this copolymer exhibited a logic-gate response to the light irradiation and to increased temperature, which has three different modes depending on the pH of the solution. Especially, a great increase in turbidity was observed even in dilute aqueous solution containing only 0.10 wt % of the copolymer although the copolymer contained spirobenzopyrans by only 1.1 mol %. It was confirmed also that the spirobenzopyran residues were isomerized by the influence of thermally induced phase transition of the same system. The main chain of thermoresponsive polymer and photoresponsive chromophores, which are linked closely together, affected each other.

An ultrathin polyion complex (PIC) layer containing β-cyclodextrin (β-CD) was formed on the surface of a charged base membrane. Thus, a positively charged copolymer containing β-CD was synthesized by the radical copolymerization of β-CD monomer and allylamine, and was used to modify the surface of a Nafion membrane containing negatively charged fixed ions. Electron spectroscopy for chemical analysis (ESCA) confirmed the presence of the copolymer on the surface of the Nafion membrane due to the formation of a PIC layer. The ability of the membrane to separate organic isomer mixtures was tested by the pervaporation technique, using butanol isomer mixtures as the feed model. The membrane exhibited a good selectivity toward butanol isomers, indicating the effectiveness of β-CD as a selective fixed carrier for the isomers.

Porous membranes composed of the biodegradable polyesters poly(D,L-lactide) (PLA) and poly(D,L-lactide-co-glycolide) (PLGA) were prepared by a phase inversion process. The molecular weights of the polymers and the concentrations of the polymer solutions affected the pore size and structure of the PLA and PLGA membranes. The molecular weights and morphological changes of the membranes as a function of time were investigated under incubation at 37°C in a humidified 5% CO2 atmosphere. The pores that formed in the membranes changed dramatically with increasing time under these conditions. From the thermal characterization of the polymers in their dry and wet states, we found that the glass-transition temperatures of PLA and PLGA affected morphological structure changes in the porous membranes. We also prepared a collagen-coated membrane to improve the interaction between the cell and the substrate, and we observed that the collagen coating enhanced the attachment and growth of Chinese hamster ovary cells on the substrate. Finally, we found that only PLA was a suitable material to prepare a porous membrane scaffold with the phase inversion process with PLA, and a collagen coating was necessary for cell culture on the membrane. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 2082–2092, 2004

CaCO3 was mineralized from solutions supersaturated only by poly(acrylic acid) (PAA), without bubbling any CO2 gas in the solution. For example, a layer of CaCO3 was built up on the surface of a chitosan membrane from a supersaturated aqueous solution containing CaCl2, Na2CO3, and PAA. In this newly developed method, the PAA alone suppresses the precipitation of CaCO3 from the bulk solution, and therefore, increases the supersaturated concentration. This concentration is estimated to be the same order as that attained in the method in which both CO2 gas and PAA were used. At the same time, PAA supplies nucleation fields by forming a polymer complex with chitosan. The crystal system obtained was different from those obtained when using CO2 gas. Self-organization of aragonite crystallites led to the formation of uniform, concentric, or branching patterns in the surface-domain structure. These patterns had morphologies similar to those discovered by other researchers, typically in the crystallization of ascorbic acid. Thicker layers of CaCO3 could be formed on chitosan membranes, the surfaces of which had been converted to a polyelectrolyte complex (PEC) by exposure to PAA solution before the onset of mineralization. Under certain conditions, the CaCO3 layer had a small spherical curvature, similar to a half-lens, and generated Newton's ring pattern from the interference fringes of visible light. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 91:3627–3634, 2004

The gas permeability of membranes composed of a polymer functionalized with azobenzene chromophores (azopolymer) was investigated under different conditions of light irradiation. A series of measurements with He gas showed that the permeability of the membranes increased by 5–10% upon irradiation with blue light, which caused cyclic photoisomerization of the azobenzene chromophores. In contrast, no significant change in gas permeability and no photoisomerization of the azobenzene chromophores were observed upon irradiation with red light. Moreover, using a digital video microscope, we observed that azopolymer irradiated with blue light spread out from localized particulate domains and thinly covered the surface of the base membrane. Together with the results of the photoresponse permeability measurements, this observation strongly suggested the existence of photoinduced plasticization as discussed in the study of photoinduced surface relief formation. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 2068–2072, 2003

Cellulose acetate (CA) membrane was modified with ultra-thin polyion complex (PIC) layers, and the pervaporation performance for water–ethanol mixture was investigated. Introduction of oxygen-containing anionic groups onto the surface of the CA membrane was attempted by the oxygen plasma treatment, and was confirmed by the electron spectroscopy for chemical analysis (ESCA). The formation of an ultra-thin PIC layer on the membrane surface by dipping of the plasma-treated membrane into an aqueous solution of polyallylamine (PALA) as the polycation was confirmed by the measurement of the attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra of the membrane. The results of the pervaporation of water–ethanol mixture showed that the PIC layer increased the selectivity of the CA membrane. The considerable increase in the selectivity was not accompanied by any large decrease in the water flux. Furthermore, it was observed that the applied energy for plasma treatment strongly affected the permeate flux and the selectivity of the CA/PIC membrane. Further improvement in the selectivity of the membrane was attained by the formation of multilayer PIC using polyacrylic acid (PAA) as the polyanion.

Poly(propylene) (PP) membrane grafted with poly(N-isopropylacrylamide) (PNIPAAm), which is known to have a lower critical solution temperature (LCST) at around 32°C, was prepared by the plasma-induced graft polymerization technique. Graft polymerization of PNIPAAm onto a PP membrane was confirmed by microscopic attenuated total reflection/Fourier transform IR spectroscopy. The grafting yield of PNIPAAm increased with the concentration of N-isopropylacrylamide monomer and the reaction time of graft polymerization. The average pore size of the PP membrane also affected the grafting yield. From the field emission scanning electron microscopy (FE-SEM) measurement, we observed a morphological change in the PP-g-PNIPAAm membrane under wet conditions at 25°C below LCST. The permeability of water through the PP-g-PNIPAAm membrane was controlled by temperature. The PP-g-PNIPAAm membrane (PN05 and PN10) exhibited higher water permeability (Lp) than the original PP substrate membrane below LCST. As the temperature increased to above LCST, Lp gradually decreased. In addition, the graft yield of PNIPAAm and the average pore size of the PP substrate influenced water permeability. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 84: 1168–1177, 2002; DOI 10.1002/app.10410

In this work, we present a novel photocleavable crosslinker to form photodegradable hydrogel through one step reaction by mixing with biocompatible polymers containing amino moieties (amino-terminated tetra-arm poly(ethylene glycol) or gelatin). Our new photocleavable crosslinker can be utilized as potentially versatile and convenient material to prepare photodegradable hydrogels with amino terminated biocompatible polymers. We present a couple of cell manipulation technique using the photodegradable hydrogels prepared with our photocleavable crosslinker.

This paper reports a simple technique to synthesize elasticity tunable hybrid hydrogels using photocleavable (N-hydroxysuccinimide terminated photocleavable tetra-arm poly(ethylene glycol); NHS-PC-4armPEG) and non-photocleavable (N-hydroxysuccinimide terminated tetra-arm poly(ethylene glycol); NHS-4armPEG) activated-ester type crosslinkers. Partially photodegradable hybrid hydrogels were synthesized by reacting the crosslinker mixture with amino-terminated tetra-arm poly(ethylene glycol) (amino-4armPEG). The photocleavable crosslinks are cleaved by irradiating light while the non-photocleavable crosslinks remain intact, resulting in decreased elasticity. We demonstrate that hydrogel elasticity can be controlled by adjusting the ratio of photocleavable NHS-PC-4armPEG and non-photocleavable NHS-4armPEG, and by varying the light exposure energy. We also show how micropatterned elasticity can be obtained in the hydrogels by irradiating with micropatterned light. These techniques could provide a novel platform to tailor the elasticity of hydrogels with microscale precision for biological studies in the near future.

Cell sorting is an essential and efficient experimental tool for the isolation and characterization of target cells. A three-dimensional environment is crucial in determining cell behavior and cell fate in biological analysis. Herein, we have applied photodegradable hydrogels to optical cell separation from a 3D environment using a computer-controlled light irradiation system. The hydrogel is composed of photocleavable tetra-arm polyethylene glycol and gelatin, which optimized cytocompatibility to adjust a composition of crosslinker and gelatin. Local light irradiation could degrade the hydrogel corresponding to the micropattern image designed on a laptop; minimum resolution of photodegradation was estimated at 20 µm. Light irradiation separated an encapsulated fluorescent microbead without any contamination of neighbor beads, even at multiple targets. Upon selective separation of target cells in the hydrogels, the separated cells have grown on another dish, resulting in pure culture. Cell encapsulation, light irradiation and degradation products exhibited negligible cytotoxicity in overall process.

Photodegradable hydrogels have emerged as powerful platforms for studying and directing cellular behavior in a spatiotemporally controlled manner. Photodegradable hydrogels have previously been formed by free radical polymerizations, Michael-type addition reactions, and orthogonal click reactions. Here, an ester-activated photocleavable crosslinker is presented for preparing photodegradable hydrogels by means of a one-step mixing reaction between the crosslinker and a biocompatible polymer containing amino moieties (amino-terminated tetra-arm poly(ethylene glycol) or gelatin). It is demonstrated that photodegradable hydrogels micropatterned by photolithography can be used to culture cells with high viability and proliferation rates. The resulting micropatterned cell-laden structures can potentially be used to create 3D biomaterials for various tissue-engineering applications.

Cell micropatterning methods with stimuli-responsive dynamic surfaces are getting a lot of attention in a wide variety of research fields, ranging from cell engineering to fundamental studies in cell biology. The surface of a slide coated with photo-cleavable poly(ethylene glycol) (PEG)–lipid can be used to spatiotemporally control cell immobilization and release by light irradiation. On the basis of this surface, it is easy to design simple methods for making a fine micropattern of any kind of cell. Furthermore, target cells can be selectively and rapidly released from this surface by light irradiation. In this review, we first describe how to obtain the photo-cleavable PEG–lipid from commercially available compounds through a facile four-step synthesis. Next, as a cell-patterning method, the protocols of coating substrates with the PEG–lipid, irradiating a pattern of light onto the coated substrate, and loading cells onto the irradiated surface are described. These protocols require no expensive equipment and potentially apply to any substrates that can adsorb serum albumin or chemically expose amine moieties on their surfaces. Finally, as an advanced method, cell release from the PEG–lipid surface in microfluidic devices is introduced. We also discuss the advantages and the possible applications of the present dynamic cell-patterning method.

Collagen gel was prepared by an ordinary method as that used for preparing a cell-culture medium from an aqueous type-1 atelocollagen solution through crosslinking with glutaraldehyde (GA). Solute diffusivity in the gels prepared in the different conditions was discussed, being related to their water content. The water content of the gel decreased with increasing GA concentration at crosslinking but the solute diffusivity in the gel hardly changed. On the other hand, collagen concentration, at crosslinking of the collagen solution, dramatically affected the solute diffusivity of the obtained gel, although the water content of the gel was not changed and maintained at higher value around 0.9. The inconsistency suggested that the gel had a heterogeneous microstructure although the content of the gel was almost water. This result suggests that the process to prepare collagen medium for cell culture affects cell growth in the case that mass transfer is dominant.

The vitrification line for the ternary system of polysulfone (PSf)/N-methyl-2-pyrrolidinone (NMP)/water was determined by differential scanning calorimeter (DSC) measurements with varying compositions. Pure PSf showed both α- and β-transition temperatures (Tg,α = 187.5°C, Tg,β = −21.4°C). The Tg,α of PSf decreased with increasing solvent concentration. The Tg,α of PSf decreased linearly with the addition of NMP in the concentration range of 70–90 wt % polymer. The vitrification line was indicated in the phase diagram for the ternary system of PSf/NMP/water at 15 and 60°C. As the temperature is increased, a high polymer concentration was needed to reach the vitrification condition. The vitrification composition of the polymer in the binary system of PSf and NMP was 72.0 wt % at 15°C and 79.8 wt % at 60°C. We also found that the slope of the vitrification line changed with the temperature and that a small amount of water (10–20 wt %) can induce the vitrification of the polymer solution in the PSf/NMP/water system at 15°C. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 71: 431–438, 1999

A cyclophane (CP44)-bonded silica gel stationary phase was prepared and elution behaviour of hydrophobic solutes was investigated in the reversed-phase mode. Aromatic compounds were retained on the stationary phases more strongly than the corresponding alicyclic compounds, as was expected by the complex-forming ability of the cyclophane. The stationary phases also showed isomer-selective separation for monomethyl- and dimethylnaphthalenes. The isomers having methyl groups at the α-position were eluted prior to those having methyl groups at the β-position, i.e., the retention order for methylnaphthalene was, α

A novel aqueous two-phase system, which exhibits a reversible photoinduced phase separation, has been developed with photochromic dextran synthesized by substituting 0.3 mol % of the hydroxyl groups with the photochromic chromophore, 6-nitrospiropyran (NSp). For an aqueous solution containing this photochromic dextran and poly(ethylene glycol), it was observed that the solution, which had been uniform in the dark, quickly separated into two phases through blue light irradiation within 1 min and returned to the former uniform state spontaneously after heating at 50 degrees C for 1 h. Photoisomerization of NSp was confirmed to shift the phase separation temperature of this aqueous two-phase system by up to 30 degrees C.

Human induced pluripotent stem cells (hiPSCs) are a promising source of cells for medical applications. Recently, the development of polydimethylsiloxane (PDMS) microdevices to control the microenvironment of hiPSCs has been extensively studied. PDMS surfaces are often treated with low-pressure air plasma to facilitate protein adsorption and cell adhesion. However, undefined molecules present in the serum and extracellular matrix used to culture cells complicate the study of cell adhesion. Here, we studied the effects of vitronectin and γ-globulin on hiPSC adhesion to plasma-treated and untreated PDMS surfaces under defined culture conditions. We chose these proteins because they have opposite properties: vitronectin mediates hiPSC attachment to hydrophilic siliceous surfaces, whereas γ-globulin is adsorbed by hydrophobic surfaces and does not mediate cell adhesion. Immunostaining showed that, when applied separately, vitronectin and γ-globulin were adsorbed by both plasma-treated and untreated PDMS surfaces. In contrast, when PDMS surfaces were exposed to a mixture of the two proteins, vitronectin was preferentially adsorbed onto plasma-treated surfaces, whereas γ-globulin was adsorbed onto untreated surfaces. Human iPSCs adhered to the vitronectin-rich plasma-treated surfaces but not to the γ-globulin-rich untreated surfaces. On the basis of these results, we used perforated masks to prepare plasma-patterned PDMS substrates, which were then used to pattern hiPSCs. The patterned hiPSCs expressed undifferentiated-cell markers and did not escape from the patterned area for at least 7 days. The patterned PDMS could be stored for up to 6 days before hiPSCs were plated. We believe that our results will be useful for the development of hiPSC microdevices.