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PURPOSE: A microwell culture system that facilitates group culture, such as well-of-the-well (WOW), improves embryonic development in an individual culture. We examined the effect of WOW on embryonic development in vitro with commercially available human single culture media. METHODS: Using four different commercial human single culture media, in vitro development and imprinted gene expression of bovine embryos cultured in WOW were compared to droplet culture (one zygote per drop). To determine the effects of microwell and group culture on embryonic development, different numbers of embryos were cultured in droplet or WOW. Diffusion simulation of accumulating metabolites was conducted using the finite volume method. RESULTS: WOW had a positive effect on bovine embryonic development, regardless of the type of single culture media. Imprinted gene expression was not different between droplet- and WOW-derived blastocysts. The microwell and group cultures in WOW showed a significant positive effect on the rate of total blastocysts and the rate of development to the expanded and hatching blastocyst stages. The assumed cumulative metabolite concentration of WOW with one embryo was 1.47 times higher than that of droplet culture with one embryo. Furthermore, the concentration of WOW with three embryos was 1.54 times higher than that of WOW with one embryo. CONCLUSIONS: In using human single culture media, a microwell culture system that allows group culture could be a powerful clinical tool for improving the success of assisted reproductive technologies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-018-1252-z) contains supplementary material, which is available to authorized users.

Conventionally, in vitro-fertilized (IVF) bovine embryos for transfer are morphologically evaluated at day 7-8 of embryo culture. This method is, however, subjective and results in unreliable selection. We previously described a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse monitoring in our specially developed microwell culture dishes (LinKID micro25). The system can noninvasively identify prognostic factors that reflect viability after transfer. By assessing a combination of identified prognostic factors -timing of the first cleavage; number of blastomeres at the end of the first cleavage; and number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle- the pregnancy rate was improved over using conventional morphological evaluation. Time-lapse monitoring with LinKID micro25 could facilitate objective and reliable selection of healthy IVF bovine embryos. Here, we review the novel bovine embryo selection system that allows for prediction of viability after transfer.

To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 μl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density.

We have developed a polystyrene-based well-of-the-well (WOW) system using injection molding to track individual embryos throughout culture using time-lapse cinematography (TLC). WOW culture of bovine embryos following in vitro fertilization was compared with conventional droplet culture (control). No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality and inner cell mass (ICM) and trophectoderm (TE) cell numbers were not different between control- and WOW-derived blastocysts; however, apoptosis in both the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at Days 30 and 60 after embryo transfer (P < 0.05). TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pickup; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of two blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.

Contents The purpose of this study was to assess the feasibility of polyester mesh culture for the in vitro production of bovine embryos, as polyester mesh is an alternative way for tracking individual embryos throughout culture using time-lapse cinematography (TLC). Bovine embryos were isolated during in vitro culture using sections of three different polyethylene terephthalate (PET) mesh products. In vitro matured and fertilized bovine oocytes were cultured in the 217 × 217, 230 × 230 or 238 × 238-μm openings of PET mesh sections or in simple micro-drops (control) for 7 days under either 20% or 5% O2 tensions. No difference in embryo developmental rates was found between the culture groups in terms of cleavage, blastocyst formation and blastocyst expansion irrespective of O2 tension. In contrast, under 20% O2 tension, blastocysts that developed in PET mesh with 217 × 217-μm opening had significantly higher numbers of total and trophectoderm (TE) cells than control embryos; however, the numbers and proportions of inner cell mass (ICM) cells did not differ. Under 5% O2 tension, no difference was found among the culture groups in the numbers of total, ICM and TE cells in embryos. All three PET mesh products investigated in this study were proven to be effective to prevent embryo movement. The results demonstrate that bovine embryos can be cultured in PET mesh sections without negative side-effects and suggest that embryo distance determined by the mesh affects embryo quality at atmospheric oxygen tension. Polyethylene terephthalate mesh with 217 × 217-μm openings was found to be the most suitable for further application in TLC.

A new series of nanotubes synthesized from α-cyclodextrin (molecular nanotube) form an inclusion complex with polyaniline, a conducting polymer. This inclusion complex can be considered to be an insulated molecular wire (IMW). In this study, we investigated the conductivity of this insulated molecular wire by using narrow-gap (150 nm) electrodes fabricated by a scanning-probe nanofabrication technique. The IMW, on doping, showed nearly ohmic I - V characteristics, and the conductivity was estimated to be 4 × 10 −2 S/cm.

We report on a simple process for fabricating fine electrodes by using shadow mask evaporation and tip-induced local oxidation. A set of electrodes for four-term resistance measurement has been fabricated. The gap width between the fine electrode was 150 nm and the roughness of the electrode surface was less than 0.5 nm. We were able to use the electrodes to measure the conductivity of a multiwalled carbon nanotube (MWNT).

Inclusion complex formation between a conducting polymer, polyaniline (PANI) with emeraldine base, and a molecular nanotube synthesized from α-cyclodextrin (α-CD) has been studied by atomic force microscopy. We observed a rodlike inclusion complex of PANI and the molecular nanotube on mica substrate at room temperature. The height of this structure is nearly equal to the outside diameter of α-CD and almost uniform along the whole length of the structure, which indicates that a conducting wire of PANI is fully covered by molecular nanotubes as insulator. Accordingly, this inclusion complex can be regarded as insulated molecular wire.

We have formed insulated molecular wires of conducting polymers, polyaniline (PANI), and cyclodextrin (CD) or the molecular nanotubes synthesized from CDs, and observed the structures of the wires by atomic force microscopy (AFM). CD or the molecular nanotube can isolate a single conducting polymer chain by shielding the attractive interaction between polymer chains. In addition, since CD or the nanotube confines the conformation of the polymer chain to a rodlike one owing to the very small cavity, the defects of the π-conjugated system should be suppressed on the polymer chain. Further four-term electrodes with gaps of the order of 100 nm have been made by two different methods using AFM, the tip-induced local oxidation and AFM lithography, in order to measure the conductivity of the insulated molecular wires. With the electrodes fabricated by the tip-induced local oxidation, it was difficult to measure the conductivity because of the native oxide layer on the electrode surface. On the other hand, the AFM lithography provided us with the electrodes appropriate for the conductivity measurements of molecular wires.

The lymphatic system in skin plays important roles in drainage of wastes and in the afferent phase of immune response. We previously showed that activation of vascular endothelial growth factor receptor (VEGFR), specifically the VEGFC/VEGFR-3 pathway, attenuates oedema and inflammation by promoting lymphangiogenesis, suggesting a protective role of lymphatic vessels against skin inflammation. However, it remains unknown how physical stimuli promote lymphatic function. Here, we show that lymphatic endothelial cells (LECs) are activated by direct-current (DC) electrical stimulation, which induced extension of actin filaments of LECs, increased calcium influx into LECs, and increased phosphorylation of p38 mitogen-activated protein kinase (MAPK). An inhibitor of focal adhesion kinase, which plays a role in cellular adhesion and motility, diminished the DC-induced extension of F-actin and abrogated p38 phosphorylation. Time-lapse imaging revealed that pulsed-DC stimulation promoted proliferation and migration of LECs. Overall, these results indicate that electro-stimulation activates lymphatic function by activating p38 MAPK.

The properties of organic electroluminescence devices are largely affected by the kind of metals used as the cathode, because the barrier height formed at the interface depends on the electronic and chemical properties of the metals. On the basis of the temperature dependence of currents flowing through the interface between tris(8‐hydroxyquinolino)aluminum and a magnesium cathode, the carrier injection mechanism at the interface was ascribed to the thermionic emission with the barrier height of about 0.58±0.03 eV at zero electric field. The barrier height became larger when metals with larger work functions were used as the cathode. The current density was also found to be dependent on the chemical structure of the interfaces.

We have fabricated four-probe Pt electrodes on a sapphire substrate by scanning-probe nanofabrication and reactive ion beam etching. Surface roughness, gap width and height of the fabricated electrodes were 0.3 nm, 150 nm and 4 to 5 nm, respectively. In order to test the quality of the fine electrodes, we evaluated the current through a multi-walled carbon nanotube (MWNT) placed on the electrodes. The measured two-probe resistance of the MWNT was 130 kΩ which included the contact resistance, while the leakage resistance was larger than 1 TΩ.

To identify embryos individually during in vitro development, we previously developed the well-of-the-well (WOW) dish, which contains 25 microwells. Here we investigated the effect of embryo density (the number of embryos per volume of medium) on in vitro development and gene expression of bovine in vitro-fertilized embryos cultured in WOW dishes. Using both conventional droplet and WOW culture formats, 5, 15, and 25 bovine embryos were cultured in 125 µl medium for 168 h. The blastocysts at Day 7 were analyzed for number of cells and expression of ten genes (CDX2, IFN-tau, PLAC8, NANOG, OCT4, SOX2, AKR1B1, ATP5A1, GLUT1 and IGF2R). In droplet culture, the rates of formation of >4-cell cleavage embryos and blastocysts were significantly lower in embryos cultured at 5 embryos per droplet than in those cultured at 15 or 25 embryos per droplet, but not in WOW culture. In both droplet and WOW culture, developmental kinetics and blastocyst cell numbers did not differ among any groups. IFN-tau expression in embryos cultured at 25 embryos per droplet was significantly higher than in those cultured at 15 embryos per droplet and in artificial insemination (AI)-derived blastocysts. Moreover, IGF2R expression was significantly lower in the 25-embryo group than in the 5-embryo group and in AI-derived blastocysts. In WOW culture, these expressions were not affected by embryo density and were similar to those in AI-derived blastocysts. These results suggest that, as compared with conventional droplet culture, in vitro development and expression of IFN-tau and IGF2R in the microwell system may be insensitive to embryo density.

We have developed a polystyrene-based well-of-the-well (WOW) system using injection molding to track individual embryos throughout culture using time-lapse cinematography (TLC). WOW culture of bovine embryos following in vitro fertilization was compared with conventional droplet culture (control). No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality and inner cell mass (ICM) and trophectoderm (TE) cell numbers were not different between control- and WOW-derived blastocysts; however, apoptosis in both the ICM and TE cells was reduced in WOW culture (P0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at Days 30 and 60 after embryo transfer (P0.05). TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pickup; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of two blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.

We have reported the formation and property of an insulated molecular wire formed by emeraldine base polyaniline (EB-PANI) and molecular nanotube. In this study, the insulated molecular wire was doped by HCl solution. The doping induced the broad absorption band around 800 nm and higher wavelength due to the polaron formation. When a solution of the insulated molecular wire was dropped and dried out on a mica substrate, the number of the insulated molecular wires attached to the mica surface was increased by the doping. This suggested that the HCI-doped cationic insulated molecular wire was strongly attached to the anionic mica surface than the non-doped insulated molecular wire. Consequently, the insulated molecular wire could he duped by HCl solution without dissociation.

We have developed a polystyrene-based well of-the-well system (WOW) using injection moulding to track individual embryos throughout culture using time-lapse cinematography (TLC). The WOW-cultured bovine embryos following in vitro fertilization (IVF) were compared with conventional droplet (control)-cultured embryos on in vitro and in vivo development. Twenty-five of zygotes were cultured in each culture system containing 125 μL of CR1aa medium supplemented with 5% calf serum for 168 h after IVF. No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality, inner cell mass (ICM), and trophectoderm (TE) cell numbers and post-vitrification survival rates were not different between control- and WOW-derived blastocysts; however, incidence of apoptosis in the ICM and TE cells was reduced in WOW culture (P

Polydimethylsiloxane (PDMS) is a non-toxic silicon compound. Its excellent optical characteristics and easy preparation make it a good candidate material for the molding of custom-shaped dishes for embryo culture. We investigated the feasibility of PDMS-based well of the well (WOW) dishes for in vitro culture of bovine embryos under different oxygen tensions. The WOW dishes with 25 micro-wells (each of 175 μm depth and 250 μm width in diameter arranged in 5 columns and 5 rows) were molded from PDMS prepared either under atmospheric (Experiment 1) or reduced (0.1 MPa) (Experiment 2) air pressure to remove air bubbles. Presumptive zygotes obtained by the in vitro maturation and fertilization of follicular oocytes were placed and cultured for 7 days in traditional micro-drops of culture medium (Control) or in the micro-wells of PDMS-based WOW dishes (PDMS-WOW), both covered by paraffin oil. The culture medium was CR1aa supplemented with 5% calf serum. The culture drop size was 125 μL (5 μL/oocyte) in both groups. Embryo development and blastocyst cell numbers between Control and PDMS-WOW groups were compared either under 20% or 5% O2 tensions. There was no statistical difference in cleavage and blastocyst rates (ranging between 82.3-86.4% and 34.0-45.8%, respectively) between Control and PDMS-WOW embryos irrespective of oxygen tension and dish production method. In Experiment 1, the mean total cell numbers in blastocysts were lower in the PDMS-WOW group than that in Control under 20% O2 (105.0 ± 5.5 and 130.4 ± 9.9, respectively) (P < 0.05, ANOVA); however, the application of 5% O2 significantly improved the cell numbers and eliminated the difference between the PDMS-WOW and Control groups (135.4 ± 6.2 and 148.0 ± 9.0, respectively). In Experiment 2, there was no significant difference in mean total cell numbers in blastocysts between the PDMS-WOW and Control either under 20% O2 (97.2 ± 5.7 and 103.9 ± 8.9, respectively) or 5% O2 (147.5 ± 12.1 and 157.3 ± 3.9, respectively). The numbers and rates of inner cell mass and trophectoderm cells did not differ between the Control and PDMS-WOW groups, irrespective of O2 tension and production method. Our results demonstrate that bovine embryos can develop to the blastocyst stage in PDMS-based WOW dishes; however, it may express detrimental effects on embryonic cell numbers, which can be neutralized by the application of low O2 tension during culture or reduced air pressure during the PDMS preparation. This work was supported by the Research and Development Program for New Bio-Industry Initiatives.

We present all wet processed Organic Light Emitting Diodes (OLED) by using polymer OLED materials and a novel paste cathode. The stability and the strong electron injection of the cathode were achieved by Ga liquid metals based alloy with Ca. and the cathode was pattern coated by using metal mask. The red emission OLED showed 100(cd/m2), 1(cd/A) at 3.2V and 2000hrs lifetime which is the brightness half degradation.

Recently, we have reported the formation of an insulated molecular wire of a conducting polymer, emeraldine base polyaniline, and a molecular nanotube synthesized from α-cyclodextrin. In this study, we manipulated the insulated molecular wire with the cantilever tip of an atomic force microscope (AFM). We found that the insulated molecular wire was moved or cut off by the manipulation process. The results of manipulation with varying AFM tip loading forces indicated that the insulated molecular wire is cut off at loading forces larger than approximately 30 nN.

The mechanism of hole injection from the indium-tin-oxide (ITO) electrode into the hole-transporting layer of the organic electroluminescent (EL) device was concluded to be Schottky emission. The barrier height at the ITO/hole-transporting layer interface was determined to be about 0.39 eV and the field-free current density to be 7.1 ×10-2 Am-2 at room temperature. From the analysis of the current–voltage (I–V) characteristics of the organic EL devices on the basis of the carrier injection mechanisms, we found that the currents of the organic EL devices having the organic bilayer structure are governed by the properties of carrier injection from the electrodes. Important information on the potential distribution in the organic layers and the effective dielectric constant for the carrier injection processes were also acquired from the comparison of the I–V curves with the theoretical calculations.

The recombination processes of electrons and holes injected into the tris(8-hydroxyquinolino)aluminum (ALQ) layer of organic electroluminescent (EL) devices having the ALQ/diamine biller structure were studied. From the comparison of the EL and PL (photoluminescence) emissions, the quantum efficiency of EL under the normal operational conditions was estimated to be about 0.12 times that of PL. Experimental results suggested the occurrence of nonemissive electron-hole recombination processes at the ALQ/diamine interface.