Magnetic self-assembly of toroidal hepatic microstructures for micro-tissue fabrication.

In this study, we developed a procedure for assembling hepatic microstructures into tube shapes using magnetic self-assembly for in vitro 3D micro-tissue fabrication. To this end, biocompatible hydrogels, which have a toroidal shape, were made using the micro-patterned electrodeposition method. Ferrite particles were used to coat the fabricated toroidal hydrogel microcapsules using a poly-L-lysine membrane. The microcapsules were then magnetized with a 3 T magnetic field, and assembled using a magnetic self-assembly process. During electrodeposition, hepatic cells were trapped inside the microcapsules, and they were cultured to construct tissue-like structures. The magnetized toroidal microstructures then automatically assembled to form tube structures. Shaking was used to enhance the assembly process, and the shaking speed was experimentally optimized to achieve the high-speed assembly of longer tube structures. The flow velocity inside the dish during shaking was measured by particle image velocimetry. Hepatic functions were evaluated to check for side-effects of the magnetized ferrite particles on the microstructures. Collectively, our findings indicated that the developed method can achieve the high-speed assembly of a large number of microstructures to form tissue-like hepatic structures.

On-Chip Fabrication of Cell-Attached Microstructures using Photo-Cross-Linkable Biodegradable Hydrogel.

We developed a procedure for fabricating movable biological cell structures using biodegradable materials on a microfluidic chip. A photo-cross-linkable biodegradable hydrogel gelatin methacrylate (GelMA) was used to fabricate arbitrary microstructure shapes under a microscope using patterned ultraviolet light. The GelMA microstructures were movable inside the microfluidic channel after applying a hydrophobic coating material. The fabricated microstructures were self-assembled inside the microfluidic chip using our method of fluid forcing. The synthesis procedure of GelMA was optimized by changing the dialysis temperature, which kept the GelMA at a suitable pH for cell culture. RLC-18 rat liver cells (Riken BioResource Research Center, Tsukuba, Japan) were cultured inside the GelMA and on the GelMA microstructures to check cell growth. The cells were then stretched for 1 day in the cell culture and grew well on the GelMA microstructures. However, they did not grow well inside the GelMA microstructures. The GelMA microstructures were partially dissolved after 4 days of cell culture because of their biodegradability after the cells were placed on the microstructures. The results indicated that the proposed procedure used to fabricate cell structures using GelMA can be used as a building block to assemble three-dimensional tissue-like cell structures in vitro inside microfluidic devices.

A novel single virus infection system reveals that influenza virus preferentially infects cells in g1 phase.

BACKGROUND: Influenza virus attaches to sialic acid residues on the surface of host cells via the hemagglutinin (HA), a glycoprotein expressed on the viral envelope, and enters into the cytoplasm by receptor-mediated endocytosis. The viral genome is released and transported in to the nucleus, where transcription and replication take place. However, cellular factors affecting the influenza virus infection such as the cell cycle remain uncharacterized. METHODS/RESULTS: To resolve the influence of cell cycle on influenza virus infection, we performed a single-virus infection analysis using optical tweezers. Using this newly developed single-virus infection system, the fluorescence-labeled influenza virus was trapped on a microchip using a laser (1064 nm) at 0.6 W, transported, and released onto individual H292 human lung epithelial cells. Interestingly, the influenza virus attached selectively to cells in the G1-phase. To clarify the molecular differences between cells in G1- and S/G2/M-phase, we performed several physical and chemical assays. Results indicated that: 1) the membranes of cells in G1-phase contained greater amounts of sialic acids (glycoproteins) than the membranes of cells in S/G2/M-phase; 2) the membrane stiffness of cells in S/G2/M-phase is more rigid than those in G1-phase by measurement using optical tweezers; and 3) S/G2/M-phase cells contained higher content of Gb3, Gb4 and GlcCer than G1-phase cells by an assay for lipid composition. CONCLUSIONS: A novel single-virus infection system was developed to characterize the difference in influenza virus susceptibility between G1- and S/G2/M-phase cells. Differences in virus binding specificity were associated with alterations in the lipid composition, sialic acid content, and membrane stiffness. This single-virus infection system will be useful for studying the infection mechanisms of other viruses.

The lipid mediator protectin D1 inhibits influenza virus replication and improves severe influenza.

Influenza A viruses are a major cause of mortality. Given the potential for future lethal pandemics, effective drugs are needed for the treatment of severe influenza such as that caused by H5N1 viruses. Using mediator lipidomics and bioactive lipid screen, we report that the omega-3 polyunsaturated fatty acid (PUFA)-derived lipid mediator protectin D1 (PD1) markedly attenuated influenza virus replication via RNA export machinery. Production of PD1 was suppressed during severe influenza and PD1 levels inversely correlated with the pathogenicity of H5N1 viruses. Suppression of PD1 was genetically mapped to 12/15-lipoxygenase activity. Importantly, PD1 treatment improved the survival and pathology of severe influenza in mice, even under conditions where known antiviral drugs fail to protect from death. These results identify the endogenous lipid mediator PD1 as an innate suppressor of influenza virus replication that protects against lethal influenza virus infection.

CXCL10-CXCR3 enhances the development of neutrophil-mediated fulminant lung injury of viral and nonviral origin.

RATIONALE: Patients who developed acute respiratory distress syndrome (ARDS) after infection with severe respiratory viruses (e.g., severe acute respiratory syndrome-coronavirus, H5N1 avian influenza virus), exhibited unusually high levels of CXCL10, which belongs to the non-ELR (glutamic-leucine-arginine) CXC chemokine superfamily. CXCL10 may not be a bystander to the severe virus infection but may directly contribute to the pathogenesis of neutrophil-mediated, excessive pulmonary inflammation. OBJECTIVES: We investigated the contribution of CXCL10 and its receptor CXCR3 axis to the pathogenesis of ARDS with nonviral and viral origins. METHODS: We induced nonviral ARDS by acid aspiration and viral ARDS by intratracheal influenza virus infection in wild-type mice and mice deficient in CXCL10, CXCR3, IFNAR1 (IFN-α/ß receptor 1), or TIR domain-containing adaptor inducing IFN-ß (TRIF). MEASUREMENTS AND MAIN RESULTS: We found that the mice lacking CXCL10 or CXCR3 demonstrated improved severity and survival of nonviral and viral ARDS, whereas mice that lack IFNAR1 did not control the severity of ARDS in vivo. The increased levels of CXCL10 in lungs with ARDS originate to a large extent from infiltrated pulmonary neutrophils, which express a unique CXCR3 receptor via TRIF. CXCL10-CXCR3 acts in an autocrine fashion on the oxidative burst and chemotaxis in the inflamed neutrophils, leading to fulminant pulmonary inflammation. CONCLUSIONS: CXCL10-CXCR3 signaling appears to be a critical factor for the exacerbation of the pathology of ARDS. Thus, the CXCL10-CXCR3 axis could represent a prime therapeutic target in the treatment of the acute phase of ARDS of nonviral and viral origins.

Culture of bovine embryos on a polydimethylsiloxane (PDMS) microwell plate.

We fabricated a polydimethylsiloxane (PDMS)-based microwell plate (PDMS-MP) containing 100 microwells with a rounded bottom and examined whether it can be used for culture of individual in vitro fertilized (IVF) embryos or parthenogenetically activated zona-free embryos in cattle. In Experiment 1, we examined the in vitro developmental ability of IVF embryos cultured individually on PDMS-MP. After IVF, 20 embryos were transferred into 100 microl drops on PDMS-MP and cultured individually in each well of PDMS-MP (PDMS group). After 7 days of culture, the embryos in the PDMS group developed to the blastocyst stage at the same rate of those in the control group cultured in a group of 20 embryos without PDMS-MP. There were no differences in total number of cells and the ratio of inner cell mass to total cells between the PDMS and control groups. In Experiment 2, we examined the in vitro developmental ability of parthenogenetically activated zona-free bovine embryos cultured individually on PDMS-MP. The zona-free embryos were cultured individually in each well of a PDMS-MP or in each well produced by pressing a darning needle onto the bottom of a culture dish (WOW group). After 7 days of culture, the blastocyst formation rate and cell number of blastocysts in the PDMS group did not differ from those of the zona-intact embryos in the control group. Also, there were no differences in the blastocyst formation rate and cell number of blastocysts between the WOW and PDMS groups. These results suggest that the culture system using PDMS-MP is useful for individual embryos or zona-free embryos in cattle.

Redescription of two Cardiodactylus species (Orthoptera, Grylloidea, Eneopterinae): the supposedly well-known C. novaeguineae (Haan, 1842), and the semi-forgotten C. guttulus ( Matsumura, 1913 ) from Japan.

In the present study, we redescribe and compare Cardiodactylus novaeguineae (Haan, 1842) and Cardiodactylus guttulus (Matsumura, 1913 ), completing previous descriptions and adding many information on morphology, including both male and female genitalia and forewing venation, distribution, habitat, behavior, calling, and courtship songs. A neotype series is selected for C. novaeguineae and deposited in RMNH, Leiden MNHN, Paris, AMNH, New York and SAMA, Adelaide. The male of C. guttulus is described, and the species C. boharti Otte, 2007 , is synonymised under C. guttulus.

On chip single-cell separation and immobilization using optical tweezers and thermosensitive hydrogel.

A novel approach appropriate for rapid separation and immobilization of a single cell by concomitantly utilizing laser manipulation and locally thermosensitive hydrogelation is proposed in this paper. We employed a single laser beam as optical tweezers for separating a target cell and locating it adjacent to a fabricated, transparent micro heater. Simultaneously, the target cell is immobilized or partially entrapped by heating the thermosensitive hydrogel with the micro heater. The state of the thermosensitive hydrogel can be switched from sol to gel and gel to sol by controlling the temperature through heating and cooling by the micro heater. After other unwanted cells are removed by the high-speed cleaning flow in the microchannel, the entrapped cell is successfully isolated. It is possible to collect the immobilized target cell for analysis or culture by switching off the micro heater and releasing the cell from the entrapment. We demonstrated that the proposed approach is feasible for rapid manipulation, immobilization, cleaning, isolation and extraction of a single cell. The experimental results are shown here.

Isolation and extraction of target microbes using thermal sol-gel transformation.

We developed a novel separation method for random screening of target microorganisms from a large heterogeneous population by using a local viscosity control. A thermal sol-gel transformation material is mixed with the sample liquid and we controlled the state from sol to gel and gel to sol reversibly based on the temperature change controlled by heating of the microelectrode with the electric current and focused laser irradiation near the target. The selected microorganisms are fixed on the bottom plate by gel, since the viscosity around the target is temporally increased by the local heating by the focused laser. The other objects are easily washed away by the cleaning flow in the microchamber. Process of fixation, cleaning, isolation and extraction of the target microbe was possible in very short time. Based on this method, two separation systems are developed and basic experimental results of fixation and isolation of targets are shown.