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We report an automated cell‐isolation system based on fluorescence image analysis of cell aggregates cultured in a photodegradable hydrogel. The system incorporates cell culture in a humidified atmosphere with controlled CO2 concentration and temperature, image acquisition and analysis, micropatterned light exposure, and cell collection by pipetting. Cell aggregates were cultured on hydrogels, and target cells were selected by phase contrast and fluorescence image analysis. After degradation of the hydrogel by exposure to micropatterned UV light, cell aggregates were transferred to a collection vessel by robotic pipetting. We assessed the system for hydrogel degradation, recovery of target cells, and contamination by off‐target cells. We demonstrated two practical applications of our method: (i) in cell aggregates from MCF‐7‐RFP strains in which 18.8% of cells produced red fluorescent protein (RFP), we successfully obtained 14 proliferative fluorescence‐positive cell aggregates from 31‐wells, and all of the isolated strains produced a higher proportion of RFP production than the original populations; (ii) after fluorescent immunostaining of human epidermal growth factor receptor 2 (HER2) in cancer cells, we successfully isolated HER2‐positive cells from a mixed population of HER2‐positive and ‐negative cells, and gene sequence analysis confirmed that the isolated cells mainly contained the target cells. [ABSTRACT FROM AUTHOR]

The phase properties of water confined in mesoporous silica MCM-41 were investigated over a temperature range of 100-298 K as a function of pore size by specific heat capacity and inelastic neutron scattering (INS) measurements. The water content of the samples was carefully controlled to ensure the capillary filled state and no overloading of water. The values of heat capacity of the pore water are higher than those of bulk ice and liquid water over the whole temperature range measured. The contribution of water in the inner part of pores (abbreviated as the internal water) was elucidated by using the heat capacity data of monolayer water measured. The entropy of the internal water was then estimated from integration of the heat capacity of the internal water. The entropy values of the internal water increase by confinement in the pores of MCM-41 in both liquid and frozen regions, indicating an increase in the deformation of the structure and/or a change in the dynamics in both regions. The INS spectra show the density of states for the librational motion of water frozen at 50 K, suggesting that the confined water is similar to amorphous ice rather than to crystalline ice. When the sample is warmed to melt, the band edge of the librational motion for water frozen in large pores (diameter of 3.6 nm) shifts to a lower energy side, indicating the weakening of intermolecular hydrogen bonds. For water in small pores (2.1 nm), on the contrary, the librational band shifts slightly to a higher energy side, suggesting the low density liquid to high density liquid transition (L-L transition) at 225-250 K. A plausible mechanism of the L-L transition of water in confinement is proposed in terms of incomplete growth of homogeneous nucleation of ice due to an interfacial free energy effect to inhibit crystallization of water confined in small pores. [ABSTRACT FROM AUTHOR]

Polymer bonding is widely employed in thermal welding, ultrasonic welding, laser welding, and surface-activated bonding. To adapt bonding properties to the smaller microstructure and complexity of a microfluidic chip, the bonding process should produce low polymer structure deformation and strong bonding strength. To fabricate a microfluidic chip without an adhesive layer, a new precision polymer bonding process that employs a nanostructure and two different layers of the same polymer material with different glass transition temperatures (Tg) is devised. Low-temperature bonding (less than 20 °C) was achieved. A deformation ratio of 1.1% was obtained, which is 10 times smaller than that for the typical thermal-bonding process. Thus, a precision bonding technique for a low-deformation microstructured chip was successfully developed. [ABSTRACT FROM AUTHOR]

The microphysiological system (MPS) is a promising tool for predicting drug disposition in humans, although limited information is available on the quantitative assessment of sequential drug metabolism in MPS and its extrapolation to humans. In the present study, we first constructed a mechanism-based pharmacokinetic model for triazolam (TRZ) and its metabolites in the entero-hepatic two-organ MPS, composed of intestinal Caco-2 and hepatic HepaRG cells, and attempted to extrapolate the kinetic information obtained with the MPS to the plasma concentration profiles in humans. In the two-organ MPS and HepaRG single culture systems, TRZ was found to be metabolized into α- and 4-hydroxytriazolam and their respective glucuronides. All these metabolites were almost completely reduced in the presence of a CYP3A inhibitor, itraconazole, confirming sequential phase I and II metabolism. Both pharmacokinetic model-dependent and -independent analyses were performed, providing consistent results regarding the metabolic activity of TRZ: clearance of glucuronidation metabolites in the two-organ MPS was higher than that in the single culture system. The plasma concentration profile of TRZ and its two hydroxy metabolites in humans was quantitatively simulated based on the pharmacokinetic model, by incorporating several scaling factors representing quantitative gaps between the MPS and humans. Thus, the present study provided the first quantitative extrapolation of sequential drug metabolism in humans by combining MPS and pharmacokinetic modeling. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Understanding tumor heterogeneity is an urgent and unmet need in cancer research. In this study, we used a morphology-based optical cell separation process to classify a heterogeneous cancer cell population into characteristic subpopulations. To classify the cell subpopulations, we assessed their morphology in hydrogel, a three-dimensional culture environment that induces morphological changes according to the characteristics of the cells (i.e., growth, migration, and invasion). We encapsulated the murine breast cancer cell line 4T1E, as a heterogeneous population that includes highly metastatic cells, in click-crosslinkable and photodegradable gelatin hydrogels, which we developed previously. We observed morphological changes within 3 days of encapsulating the cells in the hydrogel. We separated the 4T1E cell population into colony- and granular-type cells by optical separation, in which local UV-induced degradation of the photodegradable hydrogel around the target cells enabled us to collect those cells. The obtained colony- and granular-type cells were evaluated in vitro by using a spheroid assay and in vivo by means of a tumor growth and metastasis assay. The spheroid assay showed that the colony-type cells formed compact spheroids in 2 days, whereas the granular-type cells did not form spheroids. The tumor growth assay in mice revealed that the granular-type cells exhibited lower tumor growth and a different metastasis behavior compared with the colony-type cells. These results suggest that morphology-based optical cell separation is a useful technique to classify a heterogeneous cancer cell population according to its cellular characteristics. [ABSTRACT FROM AUTHOR]

Blue light-responsive photoacid generator (PAG) was pendant to poly( N-isopropylacrylamide) gel which bound on a glass plate. The gel was compressed in thickness direction by natural drying and then was irradiated with blue light. Reswelling of the gel in water was suppressed at irradiated area with a dose-dependent effect. The results were probably due to formation of additional crosslinkng of polymer network by blue light-irradiation at dry state. By using the PAG-bearing gel, a microfluidic device was fabricated in which microchannel was formed by light irradiation and subsequent water filling. [ABSTRACT FROM AUTHOR]

ABSTRACT Human induced pluripotent stem cells (hiPSCs) are a promising cell source for drug screening. For this application, self-renewal or differentiation of the cells is required, and undefined factors in the culture conditions are not desirable. Microfluidic perfusion culture allows the production of small volume cultures with precisely controlled microenvironments, and is applicable to high-throughput cellular environment screening. Here, we developed a microfluidic perfusion culture system for hiPSCs that uses a microchamber array chip under defined extracellular matrix (ECM) and culture medium conditions. By screening various ECMs we determined that fibronectin and laminin are appropriate for microfluidic devices made out of the most popular material, polydimethylsiloxane (PDMS). We found that the growth rate of hiPSCs under pressure-driven perfusion culture conditions was higher than under static culture conditions in the microchamber array. We applied our new system to self-renewal and differentiation cultures of hiPSCs, and immunocytochemical analysis showed that the state of the hiPSCs was successfully controlled. The effects of three antitumor drugs on hiPSCs were comparable between microchamber array and 96-well plates. We believe that our system will be a platform technology for future large-scale screening of fully defined conditions for differentiation cultures on integrated microfluidic devices. Biotechnol. Biotechnol. Bioeng. 2014;111: 937-947. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

As a powerful tool of cell screening and cell purification, we developed a novel method to kill adherent cells as cultured on a substrate by micro-projection of incoherent visible light. To kill the cells by the mild light irradiated by electrically controllable micro-projection systems currently available, we introduced the assist of the photo-responsive culture substrates functionalized with a photo-acid-generating polymer. In clear contrast to the existing laser-based methods requiring point scanning, areal micro-prjection of blue light with the wavelength 436 nm killed many CHO-K1 cells at a time in the irradiated area on the substrate. The effect of the photo-generated acid was so confined that selective killing of targeted cells was achieved without critical damage to the neighboring cells. Further, we demonstrated the photo-selective killing of the adherent cells after preliminarily patterning through the photo-induced removal of cell adhesion-inhibiting polymer. Biotechnol. Bioeng. 2013; 110: 348-352. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Spirobenzopyrans, which are well known as photochromic compounds, exist as thermodynamically stable protonated ring-opened isomers (protonated merocyanine form, McH) in an acidic aqueous solution in the dark. In the present study, we investigated effects of substitution of the spirobenzopyrans on a ring-opening behavior in an aqueous system. We prepared five polymerizable spirobenzopyrans that are substituted with a methoxy group or a nitro group at the 6′- or 8′-positions and without a substituent. These monomers were copolymerized with N,N-dimethylacrylamide to evaluate the spirobenzopyrans in aqueous solution. Correlation between ring-opening rates and the kind and position of the substitution can be summarized as follows: the substitution of an electron-donating methoxy group and the substitution at the 8′-position increased the ring-opening rate, whereas the substitution of an electron-withdrawing nitro group decreased the rate. The effects of the substitution can be explained by changes in the electron density of the oxygen atom of the spirobenzopyrans. [ABSTRACT FROM AUTHOR]

Prognosis for the patients with glioblastoma, the most common malignant brain tumor, remains dismal. A major barrier to progress in treatment of glioblastoma is the relative inaccessibility of tumors to chemotherapeutic agents. Convection-enhanced delivery (CED) is a direct intracranial drug infusion technique to deliver chemotherapeutic agents to the central nervous system, circumventing the blood-brain barrier and reducing systemic side effects. CED can provide wider distribution of infused agents compared to simple diffusion. We have reported that CED of a polymeric micelle carrier system could yield a clinically relevant distribution of encapsulated agents in the rat brain. Our aim was to evaluate the efficacy of CED of polymeric micellar Am80, a synthetic agonist with high affinity to nuclear retinoic acid receptor, in a rat model of glioblastoma xenografts. We also used systemic administration of temozolomide, a DNA-alkylating agent, which has been established as the standard of care for newly diagnosed malignant glioma. U87MG human glioma cells were injected into the cerebral hemisphere of nude rats. Rats bearing U87MG xenografts were treated with CED of micellar Am80 (2.4 mg/m2) on day 7 after tumor implantation. Temozolomide (200 mg/m2/day) was intraperitoneally administered daily for 5 days, starting on day 7 after tumor implantation. CED of micellar Am80 provided significantly longer survival than the control. The combination of CED of micellar Am80 and systemic administration of temozolomide provided significantly longer survival than single treatment. In conclusion, temozolomide combined with CED of micellar Am80 may be a promising method for the treatment of malignant gliomas. [ABSTRACT FROM AUTHOR]

We investigated the effect of several antiallergic agents on murine IgE-mediated biphasic cutaneous reaction. Mice were passively sensitized by an intravenous injection of monoclonal anti-dinitrophenol (anti-DNP) IgE antibody. Skin reaction was elicited by an epicutaneous challenge of dinitrofluorobenzene (DNFB) and occurred biphasically with immediate-phase response (IPR) and late-phase response (LPR) at 1 and 24 h, respectively. Classical histamine H1 receptor antagonists and some chemical mediator-release inhibitors, such as diphenhydramine and terfenadine, inhibited IPR, but not LPR. In contrast, leukotriene B[sub 4] (LTB[sub 4] ) receptor antagonist (ONO-4057) inhibited LPR only. Antagonists for LTC[sub 4] , D[sub 4] , E[sub 4] receptor (ONO-1078) and platelet-activating factor (PAF) receptor (Y-24180) significantly inhibited both IPR and LPR, similarly to prednisolone. We recently found that a third-phase inflammatory reaction with marked infiltration of eosinophils (named very-late-phase response; vLPR), which is supposed to be a more important reaction in allergic diseases, was induced, peaking at day 8 following IPR and LPR in this model. The effect of these drugs on the triphasic skin reaction can be scored based on efficacy against IPR / LPR / vLPR; +/+/+ (prednisolone, a PAF receptor antagonist Y-24180, cyclosporin A and FK-506), +/–/– (diphenhydramine), +/+/– (azelastine and LT receptor antagonist ONO-1078), and –/+/+ (an LTB[sub 4] receptor antagonist ONO-4057). Thus the inhibitory effect on vLPR as well as LPR may relate to the inhibition of eosinophil function mediated by LTB[sub 4] and PAF and/or T cells. These findings may provide the basis for a treatment modality using various antiallergic agents in allergic disease.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Engineered blood vessels generally recapitulate vascular function in vitro and can be utilized in drug discovery as a novel microphysiological system. Recently, various methods to fabricate vascular models in hydrogels have been reported to study the blood vessel functions in vitro; however, in general, it is difficult to fabricate hollow structures with a designed size and structure with a tens of micrometers scale for blood vessel tissue engineering. This study reports a method to fabricate the hollow structures in photodegradable hydrogels prepared in a microfluidic device. An infrared femtosecond pulsed laser, employed to induce photodegradation via multi-photon excitation, was scanned in the hydrogel in a program-controlled manner for fabricating the designed hollow structures. The photodegradable hydrogel was prepared by a crosslinking reaction between an azide-modified gelatin solution and a dibenzocyclooctyl-terminated photocleavable tetra-arm polyethylene glycol crosslinker solution. After assessing the composition of the photodegradable hydrogel in terms of swelling and cell adhesion, the hydrogel prepared in the microfluidic device was processed by laser scanning to fabricate linear and branched hollow structures present in it. We introduced a microsphere suspension into the fabricated structure in photodegradable hydrogels, and confirmed the fabrication of perfusable hollow structures of designed patterns via the multi-photon excitation process. [ABSTRACT FROM AUTHOR]

We have investigated the excitation wavelength dependence of solvation dynamics in the water pool of a reversed micelle using dynamic Stokes shift method. The solvation dynamics displays nanosecond and subnanosecond components, which are almost independent of the excitation wavelength. On the other hand, the total dynamics Stoke shift decreases with increasing the excitation wavelength. [ABSTRACT FROM AUTHOR]

6-Amino-6-deoxychitosan was prepared from 6-deoxy-6halo-N-phthaloylchitosan via 6-azidation. The product has high stereoregularity because of the effective and regioselective reactions. To evaluate 6-amino-6-deoxychitosan as a gene transfer agent, transfection efficiencies of its complexes with plasmid DNA in COS-1 cells were examined. [ABSTRACT FROM AUTHOR]

N-phthaloylchitosan with high structural regularity found to be a good substrate for nucleophilic substitution with N-halosuccinimides and triphenylphosphine in N-methyl-2-pyrrolidone, which provided the corresponding 6-deoxyhalogenated derivatives in a highly regioselective manner. [ABSTRACT FROM AUTHOR]

Investigates the N-acylation of chitosan by means of cyclid acid anhydride treatment in aqueous homogeneous media. Features of chitosan amino polysaccharide; Analysis of the thermal dehydration of the resulting chitosan derivatives; Percentage of sodium hydrogen carbonate used in the study.

Cell culture under medium flow has been shown to favor human brain microvascular endothelial cells function and maturation. Here a three-dimensional in vitro model of the human brain microvasculature, comprising brain microvascular endothelial cells but also astrocytes, pericytes and a collagen type I microfiber – fibrin based matrix, was cultured under continuous medium flow in a pressure driven microphysiological system (10 kPa, in 60–30 s cycles). The cells self-organized in micro-vessels perpendicular to the shear flow. Comparison with static culture showed that the resulting interstitial flow enhanced a more defined micro-vasculature network, with slightly more numerous lumens, and a higher expression of transporters, carriers and tight junction genes and proteins, essential to the blood-brain barrier functions. Image 1 • Culture under continuous interstitial flow of a 3D in vitro brain micro-vessel model. • A more defined brain microvasculature after 7 days under flow culture. • No significant change in lumen diameters, but a tendency for more numerous lumen. • Flow stress enhanced gene expression of tight junction, transporters and carriers. • Same tendency for protein expression. [ABSTRACT FROM AUTHOR]

ABSTRACT Background: In the present study, we investigated the effect of social isolation stress on IgE-mediated triphasic cutaneous reactions after 2,4-dinitrofluorobenzene (DNFB) challenge in male BALB/c mice passively sensitized with anti-dinitrophenol (DNP) IgE antibody, and examined the effects of Yokukan-san (a Kampo medicine with antipsychotic action) and a reference drug (diazepam) on the stress-enhanced cutaneous reaction. Methods/Results: In response to challenge with 0.01, 0.025 and 0.05% DNFB, triphasic skin reactions, including an immediate-phase response (IPR), a late-phase response (LPR) and a very late-phase response (vLPR) at 1 and 24 h and 8 days after antigen challenge, respectively, were increased in socially isolated mice compared with group-housed mice. Oral administration of Yokukan-san attenuated the isolation stress-exacerbated triphasic skin reactions in a dose-dependent manner, whereas it had no significant effect on cutaneous reactions in the unstressed group-housed mice. In contrast, intraperitoneal administration of diazepam, a classic benzodiazepine receptor agonist, suppressed the enhanced IPR and LPR in socially isolated mice but, surprisingly, stimulated vLPR in both stressed and unstressed mice, showing different efficacy to Yokukan-san. Moreover, the elevated locomotor activity in socially isolated mice was reduced by Yokukan-san and diazepam, while the isolation stress-induced aggressive behavior was normalized only by diazepam and not by Yokukan-san. Conclusions: The results of the present study indicate that IgE-mediated triphasic cutaneous reactions were exacerbated by social isolation stress and suggest that Yokukan-san and diazepam antagonize isolation stress-induced cutaneous reactions partly through their sedative action on social isolation stress. [ABSTRACT FROM AUTHOR]