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Here we report the perfusion culture of a multi-layered tissue composed of HepG2 cells (a human hepatoma line) in a pressure-driven microphysiological system (PD-MPS), which we developed previously as a multi-throughput perfusion culture platform. The perfusion culture of multi-layered tissue model was constructed by inserting a modified commercially available permeable membrane insert into the PD-MPS. HepG2 cells were layered on the membrane, and culture medium was perfused both through and below the membrane. The seeded density (number of cells/cm2) of the culture model is 70 times that of static culture in a conventional 35-mm culture dish. Pressure-driven circulation of the medium in our compact device (8.6 × 7.0 × 4.5 cm3), which comprised two perfusion-culture modules and a pneumatic connection port, enabled perfusion culture of two multi-layered tissues (initially 1 × 105 cells). To obtain insight into the basic functionality of the multi-layered tissues as hepatocytes, we compared albumin production and urea synthesis between perfusion cultures and static cultures. The HepG2 cells grew and secreted increasing amounts of albumin throughout 20 days of perfusion culture, whereas albumin secretion did not increase under static culture conditions. In addition, on day 20, the amount of albumin secreted by the HepG2 cells in the microfluidic device was 68% of that in the conventional culture dish, which was seeded with the same number of cells but had a 70 times larger culture area. These features of high-density culture of functioning cells in a compact device support the application of PD-MPS in single- and multi-organ MPS. [Display omitted] [ABSTRACT FROM AUTHOR]

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. [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: The objective of this study was to encapsulate two synthetic retinoids Am80 and LE540 into polymeric micelles and to control the retinoids'' release rate in vitro. Highly efficient encapsulation yields of these retinoids were obtained for micelles forming from PEG-poly(benzyl aspartate) block copolymers in the wide range of the benzyl substitution degree. The in vitro release examination for LE540 indicated very stable encapsulation of this retinoid owing to its strongly hydrophobic nature. On the other hand, Am80 exhibited a rapid release in Dulbecco''s phosphate buffer saline. An addition of a hydrophobic alkyl amine in the Am80-encapsulation process successfully led to significant retardation of the Am80 release rate. A mechanism of the retardation was considered an increase of Am80 hydrophobicity due to an ion-pairing with the alkyl amine. This paper is the first report on release control in the polymeric micelle carrier system through the ion-pairing between an encapsulated drug and an additive. [Copyright &y& Elsevier]

Abstract: A chitosan derivative, 6-amino-6-deoxy chitosan (6ACT), was galactosylated and was investigated as a gene carrier. A series of galactose-modified 6ACT (Gal-6ACT) with degrees of substitution (d.s.) ranging from 3% to 50% per pyranose were prepared by reductive alkylation with lactose. DNA retardation assays showed that the electrostatic interaction between Gal-6ACT and plasmid DNA was not changed by galactose modification up to 50% per pyranose of 6ACT. Gal-6ACT with a d.s. of 38% was bound to galactose-recognizing lectin, RCA120. A significant increase in transfection efficiency for HepG2 cells was observed at degree of substitutions ranging from 18% to 50% and at N/P values ranging from 1.5 to 2.5. Under optimum conditions, Gal-6ACT showed about 10 times higher efficiency than 6ACT. However, a slight uptake by the galactose receptors on hepatocytes was observed by flow cytometric analysis. Moreover, Gal-6ACT with a d.s. of 38% mediated efficient gene transfer into both A549 and HeLa cells lacking the galactose receptor. These results suggest that the enhancement of transfection efficiency of Gal-6ACT was not due to the increase of receptor-mediated cellular uptake. In addition, the enhanced gene transfer efficiency was not specific to the galactose modification because the efficiency of glucose-modified 6ACT for HepG2 cells was similar as that of Gal-6ACT. [Copyright &y& Elsevier]

Abstract: The C-6 positions of chitosan were successively modified in a highly regioselective manner. The starting material, N-phthaloyl-chitosan, was successfully converted into the corresponding 6-deoxy-6-halo derivatives by reaction with N-halosuccinimides and triphenylphosphine in N-methyl-2-pyrrolidone. The resulting chloride and bromide derivatives were then substituted with azido groups by reaction with sodium azide at 120 and 80°C, respectively. The azido groups were then reduced to amines via formation of the triphenylphosphinimine intermediate followed by hydrolysis using aqueous hydrazine, which also led to the removal of the N-phthaloyl groups at the C-2 positions. This sequence gave 6-amino-6-deoxy-chitosan, which, unlike chitosan, is soluble in water at neutral pH. The synthesized 6-amino-6-deoxy-chitosan derivative was evaluated as a gene carrier, and the transfection efficiency for COS-1 cells was shown to be superior to chitosan. In addition, the cytotoxicity was similar to chitosan. [Copyright &y& Elsevier]

Abstract: Excitation wavelength dependence of solvation dynamics and fluorescence anisotropy of coumarin 480 (C480) is studied using picosecond time resolved emission spectroscopy in a dimyristoyl-phosphatidylcholine (DMPC) vesicle. In the DMPC vesicle, C480 exhibits 10nm red edge excitation shift indicating the heterogeneous nature of the microenvironment. The dynamic Stokes shift (Δν) and solvation time of C480 in DMPC vesicle decreases with increase in the excitation wavelength (λ ex). The decay of the emission spectral width and fluorescence anisotropy of C480 in DMPC also depend on λ ex. [Copyright &y& Elsevier]

ABSTRACT Eosinophil penetration across the basement membrane (BM) is thought to be dependent on the degradation of membrane components. In this process, matrix metalloproteinases (MMP) appear to be primarily responsible for degradation of the BM. Matrix metalloproteinases -2 and MMP-9 degrade type IV collagen, which is a major component of the BM. In the present study, the effects of eotaxin, a selective chemoattractant for eosinophils, on the expression of MMP mRNA were examined. Incubation with chemotactically active concentrations of eotaxin for 24 h enhanced the expression of mRNA for membrane-type 1 MMP (MT1-MMP), but not of mRNA for MMP-2 and MMP-9. An increase in the protein level of MT1-MMP was also detected in the cell lysate of eotaxin-treated eosinophils. These results suggest that up-regulation of MT1-MMP expression may be involved in the eotaxin-induced penetration of eosinophils. [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]

There is an urgent need for novel methods that can accurately predict intestinal absorption of orally administered drugs in humans. This study aimed to evaluate the potential of a novel gut microphysiological system (MPS), gut MPS/Fluid3D-X, to assess the intestinal absorption of drugs in humans. The gut MPS/Fluid3D-X model was constructed using a newly developed flow-controllable and dimethylpolysiloxane-free MPS device (Fluid3D-X®). Human induced pluripotent stem cells-derived small intestinal epithelial cells were employed in this model, which exhibited key characteristics of the human absorptive epithelial cells of the small intestine, including the expression of key gene transcripts responsible for drug transport and metabolism, and the presence of dome-like protrusions in the primary intestinal epithelium under air-liquid interface culture conditions. Functional studies of transporters in the constructed model demonstrated basal-to-apical directional transport of sulfasalazine and quinidine, substrates of the active efflux transporters breast cancer resistance protein and P-glycoprotein, respectively, which were diminished by inhibitors. Furthermore, a cytochrome P450 (CYP) 3A inhibitor increased the apical-to-basal transport of midazolam, a typical CYP3A4 substrate, and reduced metabolite formation. These results suggest that gut MPS/Fluid3D-X has the potential to assess the intestinal absorption of small-molecule drugs. [ABSTRACT FROM AUTHOR]

There is an urgent need for novel methods that can accurately predict intestinal absorption of orally administered drugs in humans. This study aimed to evaluate the potential of a novel gut microphysiological system (MPS), gut MPS/Fluid3D-X, to assess the intestinal absorption of drugs in humans. The gut MPS/Fluid3D-X model was constructed using a newly developed flow-controllable and dimethylpolysiloxane-free MPS device (Fluid3D-X®). Human induced pluripotent stem cells-derived small intestinal epithelial cells were employed in this model, which exhibited key characteristics of the human absorptive epithelial cells of the small intestine, including the expression of key gene transcripts responsible for drug transport and metabolism, and the presence of dome-like protrusions in the primary intestinal epithelium under air-liquid interface culture conditions. Functional studies of transporters in the constructed model demonstrated basal-to-apical directional transport of sulfasalazine and quinidine, substrates of the active efflux transporters breast cancer resistance protein and P-glycoprotein, respectively, which were diminished by inhibitors. Furthermore, a cytochrome P450 (CYP) 3A inhibitor increased the apical-to-basal transport of midazolam, a typical CYP3A4 substrate, and reduced metabolite formation. These results suggest that gut MPS/Fluid3D-X has the potential to assess the intestinal absorption of small-molecule drugs. [ABSTRACT FROM AUTHOR]

Previous studies have reported that the mice passively sensitized with anti-dinitrophenol (DNP) IgE antibody exhibited IgE-mediated cutaneous reaction with an immediate phase response (IPR) at 1 h and a late phase response (LPR) at 24 h after the challenge of dinitrofluorobenzene (DNFB). We found that the third- phase inflammatory reaction with intense and persisting infiltration of eosinophils, named ‘very late phase reaction (vLPR)’, was induced following IPR and LPR in response to DNFB in actively and passively sensitized mice, and that the peak response of vLPR was at 8 days after the challenge. This reaction was slightly observed in non-sensitized mice. Since the accumulation of eosinophils in vLPR was markedly observed when compared with that of LPR at 24 h, the vLPR may be an important reaction in allergic diseases. The development of vLPR was partly decreased in mast cell-deficient WBB6F1-W/Wv mice and was absent in T cell-deficient BALB/c-nu/nu mice in passive sensitization. These results indicate that the vLPR in the triphasic cutaneous reaction may be mainly mediated by T cells and partially by mast cells and/or IgE antibody, and consequently lead to an intense ear swelling accompanying massive infiltration of eosinophils. [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]

Highlights: [•] Photo-induced reversible proton dissociation of spirobenzopyran in aqueous systems was analyzed quantitatively. [•] The pK a values of protonated Mc and Sp were estimated to be 6.8 and 2.6, respectively. [•] The absorbance spectra of free and protonated isomers were isolated, respectively. [•] The fractions of free and protonated isomers under irradiation and in the dark were estimated. [•] Photo-induced pH drop by more than 2 was observed in an aqueous solution. [Copyright &y& Elsevier]

A novel chitosan-based polymeric surfactant, DG-chitosan, was prepared via reductive N-alkylation of chitosan with 3-O-dodecyl-d-glucose in acetate buffer (pH 4.3, 0.1 M)-methanol in the presence of sodium cyanoborohydride (NaBH3CN). DG-chitosan was swelling in water, partly dissolvable in pyridine and DMF, and completely soluble in 0.1% aqueous acetic acid. 1H and 13C NMR spectroscopic analyses in 2% acetic acid-d4-methanol-d4 together with elemental analysis showed the degree of substitution was 27%. Formation of polymeric micelles was observed by use of pyrene as a fluorescent probe, and the critical aggregation concentration (CAC) of DG-chitosan was marked equal to 28.1 mg/L. [Copyright &y& Elsevier]

Examining intestine–liver interactions is important for achieving the desired physiological drug absorption and metabolism response in in vitro drug tests. Multi-organ microphysiological systems (MPSs) constitute promising tools for evaluating inter-organ interactions in vitro. For coculture on MPSs, normal cells are challenging to use because they require complex maintenance and careful handling. Herein, we demonstrated the potential of coculturing normal cells on MPSs in the evaluation of intestine–liver interactions. To this end, we cocultured human-induced pluripotent stem cell-derived intestinal cells and fresh human hepatocytes which were isolated from PXB mice with medium circulation in a pneumatic-pressure-driven MPS with pipette-friendly liquid-handling options. The cytochrome activity, albumin production, and liver-specific gene expressions in human hepatocytes freshly isolated from a PXB mouse were significantly upregulated via coculture with hiPS-intestinal cells. Our normal cell coculture shows the effects of the interactions between the intestine and liver that may occur in vivo. This study is the first to demonstrate the coculturing of hiPS-intestinal cells and fresh human hepatocytes on an MPS for examining pure inter-organ interactions. Normal-cell coculture using the multi-organ MPS could be pursued to explore unknown physiological mechanisms of inter-organ interactions in vitro and investigate the physiological response of new drugs. [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]

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]

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]

Cellular morphology on and in a scaffold composed of extracellular matrix generally represents the cellular phenotype. Therefore, morphology-based cell separation should be interesting method that is applicable to cell separation without staining surface markers in contrast to conventional cell separation methods (e.g., fluorescence activated cell sorting and magnetic activated cell sorting). In our previous study, we have proposed a cloning technology using a photodegradable gelatin hydrogel to separate the individual cells on and in hydrogels. To further expand the applicability of this photodegradable hydrogel culture platform, we here report an image-based cell separation system imaging cell picker for the morphology-based cell separation on a photodegradable hydrogel. We have developed the platform which enables the automated workflow of image acquisition, image processing and morphology analysis, and collection of a target cells. We have shown the performance of the morphology-based cell separation through the optimization of the critical parameters that determine the system's performance, such as (i) culture conditions, (ii) imaging conditions, and (iii) the image analysis scheme, to actually clone the cells of interest. Furthermore, we demonstrated the morphology-based cloning performance of cancer cells in the mixture of cells by automated hydrogel degradation by light irradiation and pipetting. [ABSTRACT FROM AUTHOR]

Spatial pattern formation is a critical step in embryogenesis. Bone morphogenetic protein 4 (BMP4) and its inhibitors are major factors for the formation of spatial patterns during embryogenesis. However, spatial patterning of the human embryo is unclear because of ethical issues and isotropic culture environments resulting from conventional culture dishes. Here, we utilized human pluripotent stem cells (hiPSCs) and a simple anisotropic (unidirectional perfusion) culture chamber, which creates unidirectional conditions, to measure the cell community effect. The influence of cell density on BMP4-induced differentiation was explored during static culture using a conventional culture dish. Immunostaining of the early differentiation marker SSEA-1 and the mesendoderm marker BRACHYURY revealed that high cell density suppressed differentiation, with small clusters of differentiated and undifferentiated cells formed. Addition of five-fold higher concentration of BMP4 showed similar results, suggesting that suppression was not caused by depletion of BMP4 but rather by high cell density. Quantitative RT-PCR array analysis showed that BMP4 induced multi-lineage differentiation, which was also suppressed under high-density conditions. We fabricated an elongated perfusion culture chamber, in which proteins were transported unidirectionally, and hiPSCs were cultured with BMP4. At low density, the expression was the same throughout the chamber. However, at high density, SSEA-1 and BRACHYURY were expressed only in upstream cells, suggesting that some autocrine/paracrine factors inhibited the action of BMP4 in downstream cells to form the spatial pattern. Human iPSCs cultured in a perfusion culture chamber might be useful for studying in vitro macroscopic pattern formation in human embryogenesis. [ABSTRACT FROM AUTHOR]

A novel cell processing tool to cut and section a cultured cell monolayer was implemented by the micro-projection of grid pattern onto a substrate functionalized with photo-acid-generating polymer. In adequate conditions of irradiation dosage and line width of grid pattern, Madin-Darby canine kidney cell monolayer was cut into cell clumps with similar size corresponding to the projected grid pattern. The sectioned monolayers were collected as cell clumps with the similar size from the substrate maintaining their viability enough to outgrow after subculture to the other dish. Also the influence of cutout size on the growth rate of collected cell clumps after subculture was also investigated. Further, passage of human induced pluripotent stem cells with size uniformity of the colonies was achieved by using this sectioning method without critical influence on their viability and undifferentiated state. [ABSTRACT FROM AUTHOR]

Microfluidic perfusion systems enable small-volume cell cultures under precisely controlled microenvironments, and are typically developed for cell-based high-throughput screening. However, most such systems are designed to manipulate dissociated single cells, not cell aggregates, and are thus unsuitable to induce differentiation in human induced pluripotent stem cells (hiPSCs), which is conventionally achieved by using cell aggregates to increase cell–cell interactions. We have now developed a compartmentalized microfluidic perfusion system with large flow channels to load, culture, and observe cell aggregates. Homogeneously sized cell aggregates to be loaded into the device were prepared by shredding flat hiPSC colonies into squares. These aggregates were then seeded into microchambers coated with fibronectin and bovine serum albumin (BSA) to establish adherent and floating cultures, respectively, both of which are frequently used to differentiate hiPSCs. However, the number of aggregates loaded in fibronectin-coated microchambers was much lower than in BSA-coated microchambers, suggesting that fibronectin traps cell aggregates before they reach the chambers. Accordingly, hiPSCs that reached the microchambers subsequently adhered. In contrast, BSA-coated microchambers did not allow cell aggregates to adhere, but were sufficiently deep to prevent cell aggregates from flowing out during perfusion of media. Immunostaining for markers of undifferentiated cells showed that cultures on both fibronectin- and BSA-coated microchambers were successfully established. Notably, we found that floating aggregates eventually adhered to surfaces coated with BSA upon differentiation, and that differentiation depends on the initial size of aggregates. Collectively, these results suggest that the microfluidic system is suitable for manipulating hiPSC aggregates in compartmentalized microchambers. [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]

We developed a microfluidic perfusion cell culture chip that provides three different shear stress strengths and a large cell culture area for the analysis of vascular endothelial functions. The microfluidic network was composed of shallow flow-control channels of three different depths and deep cell culture channels. The flow-control channels with high fluidic resistances created shear stress strengths ranging from 1.0 to 10.0 dyn/cm 2 in the cell culture channels. The large surface area of the culture channels enabled cultivation of a large number (approximately 6.0 × 10 5 ) of cells. We cultured human umbilical vein endothelial cells (HUVECs) and evaluated the changes in cellular morphology and gene expression in response to applied shear stress. The HUVECs were aligned in the direction of flow when exposed to a shear stress of 10.0 dyn/cm 2 . Compared with conditions of no shear stress, endothelial nitric oxide synthase mRNA expression increased by 50% and thrombomodulin mRNA expression increased by 8-fold under a shear stress of 9.5 dyn/cm 2 . [ABSTRACT FROM AUTHOR]

Abstract: Fenretinide (N-(4-hydroxyphenyl)retinamide, 4-HPR) is a synthetic retinoid with high antitumor activity against a variety of malignant cells in vitro, and is a promising candidate for cancer chemoprevention and chemotherapy. To enhance the antitumor efficacy of 4-HPR in vivo, 4-HPR were encapsulated into polymeric micelles for tumor targeting by enhanced permeability and retention effects. 4-HPR encapsulated in poly(ethylene glycol)–poly(benzyl aspartate) block copolymer micelles were prepared by the evaporation method. The mean particle size of 4-HPR encapsulated in polymeric micelles was about 173nm. After intravenous injection into tumor-bearing mice, the delivery of 4-HPR by polymeric micelles increased the blood concentration and enhanced the tumor accumulation of 4-HPR over the injection of the 4-HPR encapsulated in oil-in-water (O/W) emulsions. Tumor growth was significantly delayed following treatment by 4-HPR encapsulated in polymeric micelles, which demonstrated the improved in vivo antitumor efficacy of 4-HPR. In addition, 4-HPR encapsulated in polymeric micelles did not cause any body weight loss. These results suggest that polymeric micelles are a promising and effective carrier of 4-HPR in order to enhance tumor delivery and have potential application in the treatment of solid tumor. [Copyright &y& Elsevier]

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]

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]