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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.
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Técnicas de Cultivo de Célula , Hidrogeles , Humanos , Técnicas de Cultivo de Célula/métodos , Rayos Ultravioleta , Separación Celular/métodosRESUMEN
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.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Albúminas , Técnicas de Cultivo de Célula , Células Hep G2 , Hepatocitos , Humanos , Perfusión , UreaRESUMEN
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.
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Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas , Animales , Técnicas de Cocultivo , Evaluación Preclínica de Medicamentos , Hepatocitos/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , PresiónRESUMEN
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.
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Encéfalo/irrigación sanguínea , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Encéfalo/citología , Técnicas de Cultivo de Célula , Línea Celular , Expresión Génica , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Microvasos/citología , Microvasos/metabolismo , Proteínas de Uniones Estrechas/genética , Proteínas de Uniones Estrechas/metabolismoRESUMEN
Inside living organisms, concentration gradients dynamically change over time as biological processes progress. Therefore, methods to construct dynamic microscale concentration gradients in a spatially controlled manner are needed to provide more realistic research environments. Here, we report a novel method for the construction of dynamic microscale concentration gradients in a stepwise manner around cells in micropatterned hydrogel. In our method, cells are encapsulated in a photodegradable hydrogel formed inside a microfluidic perfusion culture device, and perfusion microchannels are then fabricated in the hydrogel by micropatterned photodegradation. The cells in the micropatterned hydrogel can then be cultured by perfusing culture medium through the fabricated microchannels. By using this method, we demonstrate the simultaneous construction of two dynamic concentration gradients, which allowed us to expose the cells encapsulated in the hydrogel to a dynamic microenvironment.
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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.
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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.
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Dispositivos Laboratorio en un Chip , Hígado/metabolismo , Técnicas Analíticas Microfluídicas , Triazolam/metabolismo , Células CACO-2 , Humanos , Cinética , Hígado/patología , Técnicas Analíticas Microfluídicas/instrumentación , Modelos Biológicos , Triazolam/sangre , Triazolam/farmacocinética , Células Tumorales CultivadasRESUMEN
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.
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Bioensayo/instrumentación , Dispositivos Laboratorio en un Chip , Adsorción , Animales , Adhesión Celular , Línea Celular , Dimetilpolisiloxanos/química , Vidrio/química , Humanos , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
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.
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Automatización de Laboratorios , Separación Celular , Forma de la Célula/fisiología , Hidrogeles/química , Reconocimiento de Normas Patrones Automatizadas/métodos , Andamios del Tejido/química , Automatización de Laboratorios/instrumentación , Automatización de Laboratorios/métodos , Separación Celular/instrumentación , Separación Celular/métodos , Células Cultivadas , Células Clonales , Matriz Extracelular/química , Gelatina/química , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Hidrogeles/efectos de la radiación , Procesamiento de Imagen Asistido por Computador , Luz , Neoplasias/patología , FotólisisRESUMEN
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.
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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.
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Proteína Morfogenética Ósea 4/farmacología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Adhesión Celular/efectos de los fármacos , Recuento de Células , Células Cultivadas , Embrión de Mamíferos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/fisiología , Células Madre Pluripotentes/efectos de los fármacos , Análisis Espacial , Propiedades de SuperficieRESUMEN
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-, ß-, or γ-CD reveals that ß-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 ß-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.
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Resinas Acrílicas/química , Benzopiranos/química , Ciclodextrinas/química , Indoles/química , Nitrocompuestos/química , Temperatura , Resinas Acrílicas/síntesis química , Deshidratación , Procesos Fotoquímicos , Espectrofotometría UltravioletaRESUMEN
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.
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Neoplasias Mamarias Experimentales/patología , Animales , Línea Celular Tumoral , Proliferación Celular , Separación Celular , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Esferoides Celulares/patologíaRESUMEN
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.
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Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes Inducidas/citología , Microfluídica/métodos , Recuento de Células , Técnicas de Cultivo de Célula/instrumentación , Diferenciación Celular , Fibronectinas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Microfluídica/instrumentaciónRESUMEN
We present a novel cell culture chip, namely, "inverting microwell array chip," for cultivation of human induced pluripotent stem cells. The chip comprises a lower hydrogel microwell array and an upper polystyrene culture surface. We demonstrate the formation of uniform cellular aggregates in the microwell array, and after inversion, a culture with controlled aggregate size and geometrical arrangement on the polystyrene surface. Here, we report effects of cell concentrations on a cultivation sequence in the chip.
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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).
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Células Endoteliales de la Vena Umbilical Humana/citología , Mecanotransducción Celular/genética , Técnicas Analíticas Microfluídicas/instrumentación , ARN Mensajero/genética , Estrés Mecánico , Recuento de Células , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Técnicas Analíticas Microfluídicas/métodos , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Perfusión , ARN Mensajero/metabolismo , Resistencia al Corte , Trombomodulina/genética , Trombomodulina/metabolismoRESUMEN
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.
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Técnicas de Cultivo de Célula/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Perfusión/instrumentación , Células Madre Pluripotentes/citología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Diseño de Equipo , Proteínas de la Matriz Extracelular , Humanos , Técnicas Analíticas Microfluídicas/métodos , Perfusión/métodosRESUMEN
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.
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Dióxido de Silicio/química , Temperatura , Termodinámica , Agua/química , Difracción de Neutrones , Tamaño de la Partícula , Porosidad , Dispersión de Radiación , Propiedades de SuperficieRESUMEN
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-projection 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.
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Ácidos/metabolismo , Muerte Celular/efectos de la radiación , Técnicas Citológicas/métodos , Procesos Fotoquímicos/efectos de la radiación , Animales , Células CHO , Adhesión Celular , Cricetinae , Cricetulus , Luz , Microscopía Confocal , Polimetil Metacrilato/metabolismo , Polimetil Metacrilato/efectos de la radiaciónRESUMEN
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.