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To establish a highly efficient method of ex vivo expansion of hematopoietic cells (HCs), three-dimensional (3D) cocultures of HCs and stromal cell lines were performed using porous polymer scaffolds. Hematopoietic cells derived from mouse fetal livers were expanded by two successive cultures without the use of exogenous cytokines, namely, 3D cultures of stromal cells (DAS 104-8 cell line) to form stromal layers within the scaffolds, and, subsequently, by cocultures of the HCs on the stromal cell layers for 2 weeks. To expand the HCs more conveniently, in some experiments the stromal layers formed within the scaffolds were frozen (3D freezing) before the cocultures, then stored and applied to the cocultures after thawing. When the HCs were cocultured on the stromal layers of the DAS 104-8 cells, primitive HCs (c-kit + and CD34 + cells) were expanded several fold during the cocultures. In contrast, the expansion of these primitive HCs was remarkably enhanced in the cocultures using the 3D frozen-thawed DAS 104-8 stromal layers (c-kit + cells > fifteenfold and CD34 + cells > thirtyfold), and these expansions were significantly higher than those without the 3D freezing. The expansions enhanced by cocultures on the 3D frozen-thawed stromal layers were also observed in the cocultures with another stromal cell line (DAS 104-4). Because 3D frozen-thawed stromal cell lines are easy to handle, 3D coculture of HCs on frozen-thawed stromal cell lines may be an effective and convenient method for expanding primitive HCs.

The high plateaus in west China may provide suitable sites for astronomical observations with theinstitute’s middle-range telescopes and possibly with larger telescopes. Under China-Japan collaborationsfor site survey in west China, we have been conducting searches for good sites and monitoring theircharacteristics over several years. As recent results of our site survey show, sites in west Tibet arerevealed with a high possibility of good astronomical observations.Weather characteristics at Gar in Ali, Tibet, show its high clear-sky ratios, especially in winter, com-parable to Mauna Kea, Hawaii. But it has some wind problem in winter, where stronger wind speeds,over 20m/sec, occur frequently even though the sky is clear. To nd calmer sites, we have conductednumerical simulations for the Ali area using the Japan Meteorological Agency NonHydrostatic Model.We have found another site, named ZoZo Hill, near Gar. We will continue to monitor the Gar site toclarify weather characteristics over the whole year and hopefully start to negotiate for site monitoring atZoZo Hill this year.Key words: international collaboration, site test, telescope site, west Tibet1. WEST TIBET AS AN IMPORTANT LONGITUDI-NAL LOCATION FOR GLOBAL ASTRONOMICALOBSERVATION NETWORKAs west China is indicating its importance in playinga role for the global astronomical observation network(Yao 2005), we have been conducting astronomical site-monitorings at three sites in west China; Karasu (Xin-jiang Uighur), Oma (Tibet) and Gar/Ali (Tibet) undera China-Japan Astronomical Collaboration since 2007(Sasaki et al., 2008; Yao et al., 2011; Sasaki et al., 2011).2. ASTRONOMICAL SITE MONITORINGSeveral astronomical site-monitoring instruments havebeen deployed at the sites. At the Oma and Gar site,cloud monitoring cameras have revealed excellent skyconditions, especially in winter. Gar has shown betterweather conditions than Oma.On the other hand, strong winds that have been ob-served at the current monitoring site, Gar, in winter mayseriously a ect the number of observable nights (Figure1), as the imaging capabilities of telescope optics wouldbe reduced due to turbulence caused by stronger windhttp://pkas.kas.orginside the telescope dome by applying the criteria forwind speed

This study's primary goal was to develop an effective ex vivo expansion method for haematopoietic cells. 3D culture of mouse bone marrow cells was performed in porous scaffolds using a sheet or cube shape. Bone marrow cells were cultured on bone marrow-derived stromal layers formed within the scaffolds and the effect of scaffold shape on the expansion of haematopoietic cells was examined. In some experiments, stromal layers within cubic scaffolds were frozen and then used to culture bone marrow cells after thawing. Results show that after comparison, total cell density and expansion of haematopoietic cells were greater in cultures using the cubic scaffold, suggesting that it was superior to the sheet-like scaffold for expanding haematopoietic cells. When cryopreserved stroma was used, it effectively supported the expansion of haematopoietic cells, and a greater expansion of haematopoietic cells [(erythroid and haematopoietic progenitor cells (HPCs)] was achieved than in cultures with stromal cells that had not been cryopreserved. Expansion of cells using cryopreserved stroma had several other advantages such as a shorter culture period than the conventional method, a stable supply of stromal cells, and ease of handling and scaling up. As a result, this is an attractive method for ex vivo expansion of haematopoietic stem cells (HSCs) and HPCs for clinical use.

Fibre Multi-Object Spectrograph (FMOS) is the first near-infrared instrument with a wide field of view capable of acquiring spectra simultaneously from up to 400 objects. It has been developed as a common-use instrument for the F=2 prime-focus of the Subaru Telescope. The field coverage of 300 diameter is achieved using a new 3-element corrector optimized in the near-infrared (0.9-1.8μm) wavelength range. Due to limited space at the prime-focus, we have had to develop a novel fibre positioner, called " Echidna" , together with two OH-airglow suppressed spectrographs. FMOS consists of three subsystems: the prime focus unit for IR, the fibre positioning system/connector units, and the two spectrographs. After full systems integration, FMOS was installed on the telescope in late 2007. Many aspects of the performance were checked through various test and engineering observations. In this paper, we present the optical and mechanical components of FMOS, and show the results of our on-sky engineering observations to date. © 2010. Astronomical Society of Japan.

To develop a tissue-engineered bioartificial liver (BAL), perfusion cultures of mouse and pig fetal liver cells (FLCs) immobilized within a three-dimensional (3D) porous scaffold were performed utilizing a packed-bed reactor system. These FLCs were cultured under different medium flow rate conditions, and the effects of the flow rates on cell growth and the hepatic functions of the FLCs were investigated. In the cultures of mouse FLCs, the medium flow suppressed cell growth and the albumin secretion activity of the FLCs, and considerably lower albumin secretion than that in the 3D stationary control cultures was obtained in the perfusion cultures. In the case of pig FLCs, cell growth was also inhibited by the medium flow, however, the cells exhibited higher tolerance to the medium flow compared with mouse FLCs. The albumin secretion activity of pig FLCs was well maintained under an extremely low flow rate condition (4.8 mm/min in the reactor), and activity higher than the 3D stationary cultures was detected at a later stage (after 20 days in the perfusion cultures). These results revealed that FLCs are quite sensitive to medium flow and an extremely low shear stress is required for the perfusion cultures of FLCs.

As a preliminary investigation to establish a cryopreservation method suited for bioartificial livers (BALs), three-dimensional (3-D) cryopreservation experiments with fibroblasts were performed, in which the cells were firstly seeded into a porous scaffold, and the scaffold containing the cells was then cryopreserved. After thawing, 65% of the initially applied cells were still attached to the scaffold, and this efficiency was significantly higher than that in the control experiments (39%), in which fibroblasts cryopreserved in a suspension were seeded into the scaffold. This higher efficiency was mainly caused by higher immobilization efficiency at the time of cell seeding (83%) than in the controls (54%). Collagen coating of the scaffold in the 3-D cryopreservation enhanced immobilization efficiency at the time of cell seeding, and 1-day precultures before the 3-D cryopreservation considerably improved cell growth after thawing. From these favorable results, this 3-D cryopreservation method may become useful for developing BALs.

For the purpose of applying fetal liver cells (FLCs) as a cell source to tissue-engineered bioartificial livers, three-dimensional (3-D) cultures of FLCs using a porous polymer scaffold, as well as monolayer cultures as a control, were simultaneously performed. To achieve efficient growth and differentiation, the FLCs were cultured in the growth medium for the first 3 weeks and then cultured in the differentiation medium for 3 more weeks. In these cultures, stimulating factors (oncostatin M (OSM), epidermal growth factor (EGF), hepatocyte growth factor (HGF), or dimethyl sulfoxide (DMSO)) were added to the media, and their effects were examined. When the growth medium containing OSM and EGF was used, EGF stimulated the growth of FLCs synergistically with OSM. For the differentiation of FLCs into mature hepatocytes, DMSO added to the differentiation medium remarkably enhanced albumin secretion in the 3-D and monolayer cultures, although HGF was effective only in the monolayer culture. Microscopic observation proved that FLCs exhibited hepatocyte-like morphology only in the media containing DMSO. In conclusion, successive supply of the growth medium containing EGF and OSM and the differentiation medium containing DMSO efficiently induced the growth of the 3-D cultured FLCs and their differentiation into mature hepatocytes.

In an attempt to develop a bioartificial liver using cultured hepatocytes, we investigated the short-term and long-term viability and metabolic functions of hepatocytes cultured in a new type of packed-bed type reactor using reticulated polyvinyl formal (PVF) resin as a supporting material. Perfusion culture experiments using this reactor, as well as monolayer cultures using conventional collagen-coated Petri dishes as control experiments, were performed. It was found that the highest density of immobilized hepatocytes attained with PVF resin was on the order of 10(7) cells/cm3 PVF and that hepatocytes cultured in this type of module for up to a week showed a sufficient level of liver-specific metabolic functions, such as ammonium metabolism, urea-N synthesis, and albumin secretion, to be comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising means to develop a bioartificial organ using hepatocytes.

Bubble blood oxygenators equipped with baffles of various types in the oxygenating column were studied. The rate of hemolysis, the volumetric coefficient for oxygen absorption into blood, and the fractional gas holdup were found to be affected mainly by the superficial gas velocity. When compared with the conventional bubble blood oxygenator without baffles, the bubble oxygenators equipped with various types of baffles (i.e., horizontal perforated baffles, radial vertical baffles, and a concentric hollow cylinder with and without horizontal perforated baffles) showed less hemolysis, larger gas holdup and higher values of the coefficient for oxygen absorption. Values of the heat transfer coefficient on the surface of the cylindrical baffle, which is useful as a built-in heat exchanger, were several times greater than those for single-phase heat transfer in conventional blood heat exchangers.

Vascular endothelial growth factor (VEGF) is crucial for the development and regeneration of the liver. However, there have been no reports about VEGF secretion by cultured fetal liver cells (FLCs). In the present study, the effects of oncostatin M (OSM), which strongly stimulates the growth and albumin secretion of FLCs, on VEGF secretion and morphological changes of long-term cultured FLCs were investigated under three-dimensional (3-D) and monolayer conditions. The cultured FLCs proliferated well and showed stable secretion of VEGF for up to 1 month under both monolayer and 3-D culture conditions. The addition of OSM to cultured cells strongly enhanced VEGF secretion. Compared with 3-D cultures, VEGF secretion per cell was higher in monolayer cultures. After 1 month in culture, the FLCs in 3-D cultures formed large aggregates like liver tissue, and FLCs also formed colonies and duct-like structures after several months of culture even under monolayer conditions. In conclusion, OSM stimulated the secretion of VEGF by cultured FLCs, which seemed to contribute to the development of a liver-like structure.

A three-dimensional (3-D) culture experiment of porcine fetal liver cells (FLCs) was performed using a porous resin substrate, for the purpose of developing a bioartificial liver. A long-term 3-D culture and monolayer culture as the control were performed for more than 1 month. To promote cell growth and maturation, human oncostatin M (OSM), the human leukemia inhibitory factor (LIF), or cortisol was added to the cultures, and the effect of each agent on cell proliferation and liver-specific cellular functions was investigated. The cell numbers in both the monolayer and 3-D cultures increased gradually with time, irrespective of the supplementation of the stimulating agents. In the monolayer culture, the albumin secretion of FLCs decreased rapidly, and scarce activity was detected from 2 weeks onward under all culture conditions tested. In the 3-D cultures, neither human OSM nor human LIF had any definite effect on the albumin secretion of FLCs. However, in the cultures with cortisol, albumin secretion was maintained for a considerably long period. These findings suggest that a bioartificial liver can be developed by culturing porcine FLCs with cortisol as the stimulant.

To examine effects of local tissue cooling on contusion-induced microvascular hemodynamics and leukocytes behavior using real-time intravital microscopy.Male Wistar rats (N = 21, 130-150 g) were randomly assigned to intensive cooling group (3 degrees C, N = 7), a moderate cooling group (27 degrees C, N = 7), or control group (37 degrees C, N = 7). Contusion was induced by dropping a plastic ball on exposed cremaster muscle. After 5 min, the cremaster muscle was superfused with a saline solution for 10 min at controlled temperature of either 3 degrees C (cooling), 27 degrees C (moderate cooling), or 37 degrees C (control). Microvascular hemodynamics (vessel internal diameter, blood flow rate and erythrocyte velocity) and leukocyte behavior (rolling and adhesion) were measured from recorded videotapes in the same venules before and after contusion, and after cooling.Cooling-induced vasoconstriction was marked at 3 degrees C and moderate at 27 degrees C compared with that at 37 degrees C. Blood flow rate and erythrocyte velocity were markedly lower at 3 degrees C compared to 37 degrees C. At 27 degrees C, erythrocyte velocity was higher than that at 37 degrees C, but blood flow rate was maintained at a level similar to that at 37 degrees C. The number of rolling and adhering leukocytes at 3 degrees C and 27 degrees C were significantly less than at 37 degrees C.Our results suggest that local tissue cooling, similar to cryotherapy, improves edema and inflammatory reaction, and may be useful for reducing inflammatory response without inhibiting blood flow after contusion.

The effects of electroacupuncture stimulation (EAS) of the hind paw and the back on the mesenteric microhemodynamics in anesthetized rats were investigated using an intravital microscope system. Red blood cell (RBC) velocity in the mesenteric arterioles was measured by the dual-sensor method developed by the authors. Electrical stimulation was applied using two acupuncture needles inserted into the skin and the underlying muscles of the hind paw and the dorsal Th13-L1 level area. The hind-paw EAS evoked intensity-dependent pressor responses and increase responses in RBC velocity in mesenteric precapillary arterioles, while the back EAS evoked depressor responses and decrease responses in RBC velocity. Heart rate showed increase responses accompanying EAS either on the hind paw or the back. The pressor responses and increase responses in RBC velocity in mesenteric precapillary arterioles accompanying the hind paw EAS were abolished by an intravenous administration of alpha-adrenergic receptor antagonist (phenoxybenzamine; POB), and the tachycardiac responses were abolished by administration of beta-adrenergic receptor antagonist (propranolol). Occasional but notable reflex vasoconstrictions in the mesenteric terminal arteriole were induced by EAS either on the hind paw or the back. These vasoconstrictive responses were not affected by the administration of POB. The present study directly demonstrated that hemodynamic changes at the level of precapillary arterioles accompanying EAS either on the hind paw or the back mainly depend on the changes of systemic arterial pressure regardless of stimulus current intensities. Moreover, the results in the present study suggest some receptors other than alpha-adrenergic receptor might be involved in the mechanism of EAS-induced vasoconstriction in the mesenteric arteriole.

An overview of the current status of the 8.2 m Subaru Telescope constructed and operated at Mauna Kea, Hawaii, by the National Astronomical Observatory of Japan is presented. The basic design concept and the verified performance of the telescope system are described. Also given are the status of the instrument package offered to the astronomical community, the status of operation, and some of the future plans. The status of the telescope reported in a number of SPIE papers as of the summer of 2002 are incorporated with some updates included as of 2004 February. However, readers are encouraged to check the most updated status of the telescope through the home page, http://subarutelescope.org/index.html, and/or the direct contact with the observatory staff., Comment: 18 pages (17 pages in published version), 29 figures (GIF format), This is the version before the galley proof

We previously reported a novel in situ observation model for microcirculation of lung metastasis from subcutaneously implanted Lewis lung cancer into mouse. Using this model, we studied the correlation of blood flow and the size of lung metastasis. It was revealed that metastatic growth and its angiogenesis are suppressed by circulating angiogenesis inhibitors, such as angiostatin or endostatin, released from primary tumor. When we removed the primary tumor, the metastasized lung cancer significantly grew faster and larger. But the blood flow per area did not increase either inside or outside of the metastatic tumor. This suggests that the growth of metastatic tumor is directly regulated not by blood flow increase but by the other effects of the circulating factors.

Porous polyvinyl formal (PVF) resin and poly(lactide-caprolactone) [P(LA/CL)] sponges were examined as three-dimensional matrices for chondroinduction of cultured bone marrow mesenchymal stem cells (MSCs). Approximately 5 x 10(5) mouse MSCs were seeded in porous PVF resin or P(LA/CL) sponges and were cultured for up to 1 month in serum-free high-glucose Dulbecco's modified Eagle's medium containing 10 ng/mL transforming growth factor-beta3 and 100 nM dexamethasone for chondroinduction. After the 1-month culture period, the PVF resin and P(LA/CL) sponges contained approximately twice the amount of glycosaminoglycans compared with the control pellet. Safranin-O staining of PVF and P(LA/CL) after 1 month of culture revealed a cartilage-like extracellular matrix containing glycosaminoglycans and collagen. When implanted into nude mice, PVF and P(LA/CL) seeded with MSCs were found to be both biocompatible and chondroinductive. These highly porous scaffolds can maintain a large number of cells in a three-dimensional structure. Both are potentially promising for the chondroinduction of bone marrow MSCs for research and clinical applications.

Recent reports have focused on the mechanisms of the action of electro-acupuncture stimulation (EAS) in the regulation of blood flow to different tissues. In the knee joint, blood flow is known to be modulated mainly by sympathetic postganglionic fibers, but recently the release or induction of nitric oxide (NO) synthesis in response to electrical stimulation has also been suggested. Therefore, a direct observation of the microcirculation is needed to further understand the mechanism by which blood flow is regulated by somatic afferent stimulation. In the present study, the effects of EAS to the vastus medialis muscle on systemic hemodynamics and the knee joint microcirculation were observed in vivo using a real-time confocal laser-scanning microscope system (CLMS). Electrical stimulation (5 mA, 0.5 ms, 5 Hz) was applied for 30 min using a pair of acupuncture needles introduced into the vastus medialis muscle. To clarify a plausible involvement of NO in the responses to EAS, the stimulus was applied either in the presence or absence of N(omega)-nitro-L-arginine methyl ester (L-NAME). Stimulation to either the muscle or the skin of the thigh after blockade of neuromuscular transmission was performed to determine the involvement of muscle contraction during EAS treatment. The changes in mean arterial pressure (MAP) and diameter of the arterioles supplying the knee joint were monitored continuously until 60 min poststimulus. Significant and persistent increases in arteriolar diameter by 26 +/- 6% and MAP by 17 +/- 2%, respectively, were observed after EAS to the muscle. Electro-acupuncture to the vastus medialis in the presence of L-NAME produced a strong decrease in diameter of the knee joint arterioles by -38 +/- 14% under the baseline with a simultaneous increase of 35 +/- 5% in MAP. EAS to the skin did not produce changes in arteriolar diameter while a slight increase in MAP by 12 +/- 6% over the baseline occurred after the stimulus. EAS to the muscle after neuromuscular blockade did not produce significant changes in diameter, while an increase in MAP by 24 +/- 8% was still observed, which facts suggest that the muscle contraction is required to produce vasodilatation. These responses suggest that a dynamic balance between the autonomic nervous system and the release of NO is the primary mechanism mediating the EAS effects on knee joint microcirculation.

White blood cells roll spontaneously in venules of intact, noninflamed rat skin. We investigated noninvasively in two experimental series which leukocyte subtypes participate in this phenomenon and the possible involvement of E-selectin. Male Lewis rats were anesthetized with sodium pentobarbital, and intravital video microscopy was performed on postcapillary venules in the nail-fold of a hind leg. In series 1 acridine yellow was infused for 15 min (50 mg per kg intravenously) to stain the leukocyte nuclei in situ. With the use of fluorescence microscopy rolling leukocytes could be classified unequivocally as polymorphonuclear (granulocytes) or monomorphonuclear (lymphocytes/monocytes) by the shape of their nucleus. Irrespective of vessel depth beneath the skin surface (25–45 µm), most identified rolling leukocytes were classified as granulocytes (72%-100%; median 89%). This percentage was independent of total rolling leukocyte flux, systemic leukocyte count, or their in vitro differentiation pattern. In series 2, rats were treated with either a synthetic, highly selective E-selectin blocking peptide or a control peptide (intravenously, 12 mg peptide per kg bolus, followed by 50 mg per kg per h). E-selectin blockade significantly reduced the leukocyte rolling level to about 50% of baseline (p

The effects of electrical stimulation of the dorsal skin area on the mesenteric arterioles were investigated in anesthetized rats by the use of an intravital microscope-television system. Changes in the diameter of the mesenteric precapillary arterioles (10-40 microm in diameter) were measured with an image processor. Blood flow velocity in the mesenteric precapillary arterioles was monitored by the dual sensor method developed by the authors. Electrical stimulation was performed through two platinum electrodes placed at the right dorsal Th5-12 level skin area by the use of an electrical stimulator (0.2 ms, 20 Hz). Continuous stimulation lasting for 30 s (1-10 mA) and intermittent stimulation lasting for 10 min (3 mA) were applied. The pressor response following the depressor response was induced by a stimulus current above 8 mA. The decrease in mesenteric blood flow velocity was induced by stimulus current above 10 mA. These responses were abolished by lidocaine injection into the subcutaneous area where the electrodes were attached. No significant change in arteriolar diameter or heart rate were induced by the stimulation for 30 s. Electrical stimulation of the skin for 10 min evoked a decrease in the diameter of arterioles (-3.4 +/- 2%, p0.01, n = 12). In the adrenalectomized group, electrical stimulation of the skin for 10 min elicited a slight increase in the diameter (1.1 +/- 0.5%, n = 6). It is therefore suggested that electrical stimulation of the skin for 30 s reflexly evoked decreases in MAP and in blood flow velocity, and that the constriction of the mesenteric precapillary arterioles induced by the stimulation for 10 min was mediated by humoral adrenaline and noradrenaline released by somato-adrenal medullary reflex.

The effects of somatosensory nociceptive pinch stimulation of the hindpaw on mesenteric microvascular hemodynamics and systemic circulatory parameters were investigated in anesthetized rats using an intravital microscope-television system. Blood flow velocity in the terminal (18-40 microm in diameter) or precapillary (10-20 microm in diameter) arterioles of the mesentery was monitored by the dual sensor method developed by the authors. In the proximal terminal arterioles, blood flow velocity decreased substantially along with arteriolar constriction induced by pinching of the hindpaw for 30 s. In the distal terminal arterioles and precapillary arterioles, blood flow velocity increased after pinching. In the proximal terminal arterioles, the decrease of velocity in response to reflex vasoconstriction was abolished by intravenous injection of an alpha-blocker (phentolamine, 10 mg/kg). The increase in mesenteric precapillary arteriolar blood flow velocity (43+/-9%, p < 0.01) associated with the increase in mean arterial pressure (MAP) (22+/-1%, p < 0.01) was observed within a few seconds after the onset of the stimulation, and then the response in blood flow velocity returned to the baseline ahead of MAP response recovery after the end of the stimulus. These responses were diminished by alpha-adrenergic receptor blockade. The heart rate (HR) increase (4+/-1%, p < 0.01) induced by pinching was abolished by beta-adrenergicreceptor blockade (propranolol, 3 mg/kg, i.v.). There was a strong correlation between the increase in MAP and the decrease in renal blood flow measured by laser Doppler flowmeter (r = 0.87-0.98). Pinch stimulation of the rat hindpaw evoked changes in mesenteric arteriolar blood flow velocity that were mediated via the somato-sympathetic reflex vasoconstriction and the pressor response.

UNLABELLED Xenon may increase cerebral blood flow and intracranial pressure (ICP). To evaluate the effects of xenon on brain circulation, we measured pial vessel diameter changes, CO(2) reactivity, and ICP during xenon inhalation in rabbits. Minimum alveolar anesthetic concentration (MAC) for xenon was established in rabbits (n = 6). By using a cranial window model, pial vessel diameters were measured at 30% and 60% xenon inhalation and in time control groups (n = 15). ICP, mean arterial blood pressure, and heart rate were recorded during 30% and 60% xenon inhalation (n = 5). Pial vessel diameters were measured during hypocapnia and hypercapnia conditions in 60% Xenon and Control groups (n = 14). MAC for xenon was 85%. Xenon (0.35 and 0.7 MAC) dilated the arterioles (10% and 18%, respectively) and venules (2% and 4%, respectively) (P < 0.05). Dilation of arterioles was more prominent than that of venules. ICP, mean arterial blood pressure, and heart rate did not change during xenon inhalation. No difference in CO(2) reactivity was observed between Xenon and Control groups (P = 0.79). Sixty percent xenon (0.7 MAC) dilated brain vessels, but venule changes were small. Xenon did not increase ICP and preserved CO(2) reactivity of the brain vessels. IMPLICATIONS Xenon might increase cerebral blood flow; however, 0.7 minimum alveolar anesthetic concentration xenon preserved both low intracranial pressure and CO(2) reactivity of the cerebral vessels in the normal rabbit.

Cell seeding is one of the key procedures in the construction of tissue-engineered organs. In our previous efforts to create a bioartificial liver, high-density cultures of hepatocytes (>1 x 10(7) cells/1 cm(3)-substrate) and long-term maintenance of metabolic function were achieved with a packed-bed reactor utilizing porous poly(vinyl formal) (PVF) resin as a scaffold. However, a low seeding efficiency of about 30% remains a major obstacle to the scaleup of the reactor. In the present study, a new cell seeding method, centrifugal cell immobilization (CCI), which is based on alternating centrifugation and resuspension, was used to achieve high-density seeding and improve the seeding efficiency. Using the CCI method, the maximum density of the immobilized hepatocytes reached 3.8 x 10(7) cells/1 cm(3)-PVF, and the seeding efficiency was improved to about 43% after a relatively short immobilization process (about 15 min). Moreover, further improvement of the seeding efficiency was obtained by serial immobilization procedures. Thus, we concluded that this method is useful and effective for seeding cells into 3-dimensional scaffolds.

High-density, large-scale culture of hepatocytes is a key requirement in the development of a bioartificial liver that can replace liver functions in patients with severe liver insufficiency. We have applied a porous polymer, polyvinyl formal (PVF) resin, as a cell-supporting material for hepatocyte culture. We evaluated the performance of the culture system using PVF resin under three different culture conditions: a shake culture on conventional dishes, perfusion culture with sheet-shaped PVF, and a packed-bed-type module. Among them, the packed-bed reactor using PVF resin enabled high-density culture of hepatocytes (2×107 cells/cm3-PVF). The hepatocytes immobilized in the PVF resin maintained satisfactory metabolic functions (ammonium metabolism and albumin secretion) comparable to those of the monolayer dish cultures. Furthermore, by maintaining dissolved oxygen concentration at a relatively high level (260–460μM), the metabolic functions of the hepatocytes were improved. It was concluded that the packed-bed reactor using PVF resin is a promising system for developing a bioartificial liver using hepatocytes.

We developed a new system which enables direct observation of platelet adhesion on opaque biomaterials under shear flow conditions, by combining a thin quartz cone which produces laminar shear flows, with an upright epifluorescence microscope which visualizes stained platelets through the rotating cone. This is the first report on the observation of platelets adhered to opaque biomaterials in real time under shear flow conditions. The direct observation of platelet adhesion to expanded polytetrafluoroethylene (ePTFE) as an opaque biomaterial revealed that the kinetics of platelet adhesion to ePTFE depended greatly on shear stresses, showing that the shear stress of 5.0 dyne/cm2 induced higher adhesiveness of platelets to ePTFE than that of either 0.1 or 15 dyne/cm2. The observation also showed a difference in platelet adhesiveness among ePTFEs with different fibril lengths--0, 3.2, 18, and 35 microm--indicating that ePTFEs with shorter fibril length had lower adhesiveness of platelets under a shear stress of 5.0 dyne/cm2. It is indispensable for analyzing the phenomena of platelet adhesion to opaque biomaterials to observe in real-time rolling, adhesion, and detachment of platelets under shear stresses without disturbing shear flow conditions. The results showed that the mechanical and optical design of the system could serve this purpose.

A packed-bed reactor using reticulated polyvinyl formal (PVF) resin as a support material is a useful configuration to achieve high density culture of hepatocytes for use as a bioartificial liver. The authors investigated the effects of oxygen concentrations of the culture medium on the metabolic performance of hepatocytes cultured in the reactor. A packed-bed reactor loaded with 250 PVF resin cubes (2 x 2 x 2 mm) was used. Hepatocytes obtained from male Wistar rats were inoculated into the reactor. Culture medium was perfused from the reservoir into the reactor through an oxygenator using a roller pump. Concentration of the dissolved oxygen in the medium was controlled by changing the gas mixture ratio supplied to the oxygenator. Hepatocytes cultured in the packed-bed reactor (cell density: 8.6 x 10(6) cells/cm3 PVF) under conditions of high dissolved oxygen concentrations ranging from 260 to 460 micromol/L showed 30% higher ammonium metabolic activity and 85% higher albumin secretion activity compared with those from the monolayer culture in the earlier culture stage (up to 2 days). However, low oxygen concentrations in the medium (

The dietary effect of 1,3-biseicosapentaenoyl-2-gamma-linolenoyl glycerol (STG) on the fatty acid composition of guinea pigs was examined and compared with that of an eicosapentaenoic acid ethyl ester (EPA-E) and of a soybean oil (SBO) diet. In terms of content of plasma lipid, EPA-E had a greater hypolipidemic effect than STG. On the other hand, in terms of EPA incorporation, contents of EPA in liver lipid were almost the same in the STG and EPA-E groups. Considering that the amount of EPA administered in the EPA-E group was almost 1.5 times that of the STG group, EPA may be absorbed more effectively as the glycerol ester than as the ethyl ester in guinea pigs. In all the tissue lipids, the STG group had a higher unsaturation index (UI) than the EPA-E group even though there is a lower UI in the STG diet than the EPA-E diet. These results suggest that greater amounts of desaturase products as a whole were synthesized in the STG group than in the other two groups. The dihomo-gamma-linolenic acid/arachidonic acid (DGLA/AA) ratio in plasma total lipids in the STG group was 3.5 times that of SBO group, and the DGLA/AA ratio in the EPA-E group was half that of the SBO group. In liver lipid, the ratios of DGLA/AA and EPA/AA in the STG group were 0.687 and 0.488 (phosphatidylcholine fraction) and 0.237 and 0.752 (phosphatidylethanolamine fraction), respectively. The ratio of DGLA/AA as well as the high EPA/AA ratio obtained in the present study with the STG diet may lead to physiological alterations, including enhanced synthesis of 1- and 3-series eicosanoids.

Intraluminal resistance to gas transport between the microcirculation and tissue was neglected for a half-century following the early work of Krogh. In recent years it has come to be understood that this neglect is seriously in error. This paper reviews the background for the long period of misdirection, and progress in placing the simulation of gas transport processes on a more accurate, quantitative basis.

Changes in shape and cytoskeletal structure of vascular endothelial cells induced by fluid-imposed shear stress were studied in vivo and in vitro. In in vivo experiments, aortic tissue specimens including the flow dividers of the branching of the left subclavian artery and the aortic intercostal ostium were obtained and their endothelial cell shapes were observed using the scanning electron microscope. It was found that the shape and orientation of endothelial cells were strongly affected by blood flow conditions. In in vitro cell culture experiments, porcine aortic endothelial cells were cultured on glass coverslips and extracellular matrix coated (ECM coated) glass coverslips and exposed to shear stress using a parallel plate flow chamber. When we applied a shear stress of 2 Pa for 24 hrs, the rearrangement of F-actin filaments occurred within 3 hrs and preceded the cell shape change in the early stage after shear exposure. The endothelial cells on ECM-coated coverslips exhibited more elongated cell shapes even under no-flow conditions. After exposure to shear stress, the endothelial cells on ECM-coated glass showed more retarded elongation and orientation to the direction of flow than those on no-coated glass, suggesting that the anchorage to the substrate was enhanced by ECMs.

In order to obrain sound criteria for the optimal design and improvement of the hybrid-type artificial liver support system (ALSS) utilizing isolated hepatocytes, theoretical and experimental evaluations of the performance of the ALSS were made based on the compartmental mass transfer model and reaction kinetics of cultured hepatocytes. Rate constants of the viability and metabolic reactions of the cultured hepatocytes were determined by batchwise suspension culture experiments, and the optimal conditions for hepatocyte culture were found to be in a relatively low range of dissolved oxygen tension in the culture medium. Results of in vitro and ex vivo perfusion experiments using a hollow-fiber type module or a rotating-disk type module indicated that experimentally obtained performances of the module in terms of hepatocyte viability and a few metabolic functions were well correlated with the theoretically estimated performances. Stimulation studies related to the design problems of a full-scale device revealed that the performance of the ALSS is inevitably limited by the viability of the hepatocyte. It was suggested that a considerable improvement in the performance of ALSS is to be attained by improving hepatocyte culture conditions.

We developed a new method for visualizing the flow behavior of erythrocytes in living microvessels by the fluorescent labeling technique. Intravital microscopic observation of the microcirculation in the rat mesentery using this technique enabled quantitative analyses of a few hydrodynamic aspects of the two-phase blood flow; e.g. diameter-flow relationships in a consecutively branched vascular network and the distribution of erythrocytes in an arteriolar bifurcation.

Effects of extracellular matrices (ECMs) and fluid-imposed shear stress on the cell shape and the cytoskeletal structure of microfilaments were studied in cultured porcine aortic endothelial cells (PAECs). The PAECs were cultured until confluent on non-coated or on ECM-coated glass coverslips. The components of ECM used were type IV collagen, heparan sulfate, chondroitin sulfate and dermatan sulfate. The PAECs cultured on the mixed ECMs showed marked elongation and segmental orientation with randomly distributed cell axis even under a no-flow static condition, and the microfilaments were mostly observed in parallel with the cell axis. After shear flow exposure (2 Pa, 24-48 hrs), the PAECs on non-coated glass were significantly elongated and oriented to the flow direction, however the PAECs on ECM-coated glass showed more retarded responses than the ones on non-coated glass, indicating that the anchorage to the substrate was enhanced by ECMs. The stress fibers were reorganized in accordance with the cell shape and oriented to the flow direction. These findings suggest that ECM may act together with shear stress to modify and maintain the endothelial cell configuration.

To evaluate the effects of exercise on aortic wall elasticity and elastic components, young male rats underwent various exercise regimes for 16 weeks. In the exercised rats, the aortic incremental elastic modulus decreased significantly when under physiological strain. The aortic content of elastin increased significantly and the calcium content of elastin decreased significantly in the exercised group. The accumulated data from the exercised and sedentary groups revealed that the elastin calcium content was related positively to the incremental elastic modulus. We concluded that physical exercise from an early age decreases the calcium deposit in aortic wall elastin and that this effect probably produced in the exercised rats a distensible aorta.

This study's primary goal was to develop an effective ex vivo expansion method for haematopoietic cells. 3D culture of mouse bone marrow cells was performed in porous scaffolds using a sheet or cube shape. Bone marrow cells were cultured on bone marrow-derived stromal layers formed within the scaffolds and the effect of scaffold shape on the expansion of haematopoietic cells was examined. In some experiments, stromal layers within cubic scaffolds were frozen and then used to culture bone marrow cells after thawing. Results show that after comparison, total cell density and expansion of haematopoietic cells were greater in cultures using the cubic scaffold, suggesting that it was superior to the sheet-like scaffold for expanding haematopoietic cells. When cryopreserved stroma was used, it effectively supported the expansion of haematopoietic cells, and a greater expansion of haematopoietic cells [(erythroid and haematopoietic progenitor cells (HPCs)] was achieved than in cultures with stromal cells that had not been cryopreserved. Expansion of cells using cryopreserved stroma had several other advantages such as a shorter culture period than the conventional method, a stable supply of stromal cells, and ease of handling and scaling up. As a result, this is an attractive method for ex vivo expansion of haematopoietic stem cells (HSCs) and HPCs for clinical use.

To establish an ex vivo expansion method of haematopoietic progenitor cells (HPCs) and erythroid cells, three-dimensional (3D) cultures of mouse bone marrow cells were performed, employing a porous polyvinyl formal (PVF) resin as a scaffold. In these cultures, the effects of oxygen concentration and co-cultures with stromal cells on the expansion of HPCs and erythroid cells were investigated. When bone marrow cells were cultured under 3D conditions, HPCs and erythroid cells expanded without supplementation of exogenous cytokines, irrespective of the presence of stromal cells. On the contrary, slight expansion of HPCs or erythroid cells was observed in monolayer cultures as controls, indicating that the 3D cultures using the PVF scaffold were far better in expanding HPCs and erythroid cells than the monolayer cultures. Under hypoxic conditions, bone marrow stromal cells allowed for a 3D culture of erythroid cells and HPCs at higher cell densities compared to cultures without stromal cells, and the duration of the expansion of HPCs and erythroid cells after initiating the 3D co-cultures was prolonged. The number of these cells increased throughout the culture period up to 3 weeks under hypoxic conditions, although the number decreased after 2 weeks under normoxic conditions. In conclusion, the 3D co-culture method of haematopoietic cells with stromal cells under hypoxic conditions was confirmed to be effective in expanding HPCs and erythroid cells, and this method seemed to be useful for developing an ex vivo expansion method for haematopoietic cells. Copyright © 2010 John Wiley & Sons, Ltd.

We evaluated depth of subsurface damage on a ground surface of the ultra low expansion glass-ceramics CLEARCERAMR®-Z HS (CC-Z HS) by Ohara Inc., which is one of the candidates for material for segmented mirrors of the Thirty Meter Telescope. We made polishing spots of Magnetorheological Finishing on the ground surface of CC-Z HS and measured exposed subsurface damage features on the spot surface. We also studied on hydrofluoric acid etching of the CC-Z HS ground surface, which is expected to be an effective method to remove a subsurface damage layer compared with time-consuming polishing. We etched small ground surfaces of CC-Z HS and evaluated its uniformity.

As a preliminary investigation to establish a cryopreservation method suited for bioartificial livers (BALs), three-dimensional (3-D) cryopreservation experiments with fibroblasts were performed, in which the cells were firstly seeded into a porous scaffold, and the scaffold containing the cells was then cryopreserved. After thawing, 65% of the initially applied cells were still attached to the scaffold, and this efficiency was significantly higher than that in the control experiments (39%), in which fibroblasts cryopreserved in a suspension were seeded into the scaffold. This higher efficiency was mainly caused by higher immobilization efficiency at the time of cell seeding (83%) than in the controls (54%). Collagen coating of the scaffold in the 3-D cryopreservation enhanced immobilization efficiency at the time of cell seeding, and 1-day precultures before the 3-D cryopreservation considerably improved cell growth after thawing. From these favorable results, this 3-D cryopreservation method may become useful for developing BALs.

To enable high density culture of hepatocytes for use as a hybrid artificial liver support system or a bioreactor system, a packed-bed reactor using collagen-coated reticulated polyvinyl formal (PVF) resin was applied to a primary culture of hepatocytes. Cubic PVF resins (2 x 2 x 2 mm, mean pore size: 100, 250 or 500 microns) were used as supporting substrates to immobilize hepatocytes. Two hundred and fifty cubes were packed in a cylindrical column, and 2.6-11.3 x 10(7) hepatocytes were seeded in the column by irrigating with 3 ml of the medium containing hepatocytes. Perfusion culture experiments using this packed-bed reactor, as well as monolayer cultures using conventional collagen-coated petri dishes as control experiments, were performed. Sufficient amounts of hepatocytes were found to be immobilized in the reticulated structure of the PVF resins. The highest density of immobilized hepatocytes attained with PVF resin was 1.2 x 10(7) cells/cm3 PVF, which showed levels of ammonium removal and urea-N secretion comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising process for developing a bioreactor or a bioartificial organ using hepatocytes.

An intravital fluorescence microscope system was used to investigate the pharmacological effects of endothelin-1 (ET-1) on the coronary microcirculation in the isolated beating hearts of rats. The heart was perfused by retrograde aortic steady flow with an oxygenated Krebs-Ringer solution containing FITC-dextran. Changes in diameters of coronary microvessels accompanying the cumulative injection of ET-1 in the perfusate were observed and recorded with a video camera system. Coronary perfusion pressure was also measured during each experiment. Bolus injections of ET-1 (1-300 pmole) elicited a dose-dependent increase in perfusion pressure from 54 +/- 6 mm Hg (mean +/- SEM; n = 10, before the ET-1 injection) to 144 +/- 9 mm Hg (n = 8, at the ET-1 dose of 300 pmole). A dose-dependent narrowing of microvessels was also observed. This vasoconstriction was especially prominent in small-sized arterioles; the maximum vasoconstriction of the smaller arterioles was significantly higher than that of the larger arterioles (P less than 0.05). The response induced by ET-1 dose of 3-10 pmole was significantly larger in arterioles than in postcapillary venules in the diameter range between 10 and 40 microns. The vasoconstriction produced by ET-1 was inhomogeneous. Some part of bifurcations of arterioles showed a prominent localized vasoconstriction, and occasionally showed a complete luminal obstruction. Such a segmental vasospasm might be attributed to localized sensitivities of arterioles to ET-1. These findings suggest that ET-1 may have an important role in governing the coronary resistance and regulating the capillary flow in the myocardium.

The viscoelastic deformation of porcine aortic endothelial cells grown under static culture conditions was measured using the micropipette technique. Experiments were conducted both for control cells (mechanically or trypsin detached from the substrate) and for cells in which cytoskeletal elements were disrupted by cytochalasin B or colchicine. The time course of the aspirated length into the pipette was measured after applying a stepwise increase in aspiration pressure. To analyze the data, a standard linear viscoelastic half-space model of the endothelial cell was used. The aspirated length was expressed as an exponential function of time. The actin microfilaments were found to be the major cytoskeletal component determining the viscoelastic response of endothelial cells grown in static culture.

Diet including mold oil from a lipid accumulative fungus, containing gamma-linolenic acid, showed an inhibitory effect on thrombus formation in the microvessels of rats by the light/dye method of the authors. Male Wistar rats were fed for 3 to 4 weeks with two series of experimental diets and were examined for thrombus formation. The thrombus formation times to totally occlude, ts, were 347 sec for (mold + soybean)-oil and 236 sec for (palm + soybean)-oil in the first series of diets and 1288 sec for mold oil, 538 sec for olive oil and 575 sec for safflower oil in the second series of diets. Fatty acid composition of plasma, erythrocyte and liver lipids showed an increase in arachidonate content with the diet including the mold oil. Higher arachidonate content seem favorable in inhibiting thrombus formation with increasing PGI2 formation. In terms of the level of lipid hydroperoxides, indicated as a desaturation index of constituent fatty acids, the higher desaturation index with safflower oil gave shorter ts, which suggested some oxygen derived free radicals from polyunsaturated fatty acids were involved in the mechanism of thrombogenesis study by this method.

The role of hemodynamics on platelet thrombus formation was studied in venules and arterioles of the rat mesentery. Thrombus formation was induced by the fluorescent dye/light method for examination of the following factors: 1) the effect of wall shear rate on thrombus initiation, 2) the effect of wall shear rate on the growth of thrombi, and 3) the relation between platelet thrombus initiation and intraluminal velocity profile. The range of wall shear rate was up to approximately 1,000 1/sec in venules and from 640 to 2,900 1/sec in arterioles. Platelet thrombus initiation occurred more rapidly at higher wall shear rate in venules and at lower wall shear rate in arterioles. Thrombus initiation time was shortest around a wall shear rate of 900 1/sec in venules and around 700 1/sec in arterioles. Thrombus growth rate in venules was greatest at a wall shear rate of 1,500-2,000 1/sec. Thrombus initiation and its relation to blood flow was also examined in branched and curved microvessels. In these vessels platelet thrombi were also first initiated at the sites of higher wall shear rate in venules and of lower wall shear rate in arterioles.

The high plateaus in west China (Tibet) may provide good candidate sites possibly for ELT projects. According to satellite weather data, we found that a certain area in Tibet shows potentiality for good astronomical observations with less cloud coverage. We have explored through west Tibet to watch its topography in summer, 2004. We reanalyze meteorological data collected by GAME-Tibet project. We have started weather monitor in two candidate sites in west China; Oma in western area of Tibet and Karasu near the western boundary of China. Monitoring observations using modern astronomical site-testing techniques such as a DIMM and an IR cloud monitor camera will be started to catch up continuous monitoring of seeing and cloud coverage.

The Subaru telescope had its astronomical first light in January 1999 and has been stably operated since the common use started in December 2000. The telescope is mounted on an alt-azimuth structure. The structure of 550 tons is supported by six hydrostatic oil pads which lift the structure by 50 microns. The azimuth (Az) and elevation (El) axes are driven by direct-drive linear motors, ensuring very smooth pointing and tracking operations. The Az rail consists of eight circular arc pieces. They were installed in January 1997 with a peak-to-peak level of within 0.1mm. However at a later time, vertical undulations of the Az rail were found to be more than 0.2 mm peak-to-peak at some locations where the telescope structure in the rest position applies load. Open-loop tracking accuracy of the telescope, which was about 2 arcsec RMS on the sky, was found to be due to the undulations of the Az rail. We made a table to correct telescope pointings due to the undulations. It has made open-loop tracking accuracy better than 0.2arcsec RMS. Since then, we have been monitoring the flatness of the Az rail. So far the undulations have not changed.

This review focused on a few methodologies which the author, with a background of chemical engineering, has developed in the physiological studies of microcirculation. (1) Fluorescent tracers to visualize mass transfer and hemodynamics: By means of a high sensitive SIT camera equipped in an intravital microscope system, dynamic processes of the permeation of a fluorescent dye from the microvessels through the extravascular space to lymphatics was made to be visualized. Dynamic behaviors of the formed elements were also quantitatively analyzed by the selective fluorescent labeling technique. (2) The dye/light method to induce platelet thrombus in vivo: Intravascular platelet aggregation and subsequent thrombus formation leading to the complete occlusion of the vessels were produced in the microvasculature by the irradiation of filtered light in combination with the intravascular administration of sodium fluorescein. This method enables quantitative evaluation of thrombus formation process in terms of thrombus formation times. Effects of hemodynamic parameters on thrombogenesis in vivo were quantitatively analyzed. (3) Establishment of peritoneal disseminated tumor model: Colon tumor cells (RCN-9) were inoculated into the peritoneal cavity of male Fischer rats, and the intravital microscopic observation of angiogenic vascular growth accompanying tumor growth was made possible. Dynamic behavior of leukocytes in the microcirculation of solid tumor tissue was visualized using a fluorescent labeling technique combined with the use of a real-time confocal laser-scanning microscope.

Neointimal hyperplasia influenced by intravascular hemodynamics is considered partly responsible for restenosis after endovascular stenting. To evaluate the effect of stent configuration on fluid flow behavior, we visualized flow near stents, and measured the proliferation of cultured endothelial cells (ECs). A single-coil stent (coil pitch; CP = 2.5, 5, or 10 mm) was inserted into a glass tube and perfused at 30-90 ml/min, while the flow pattern was determined by particle imaging velocimetry. The reduction of the flow velocity near the wall was correlated with the decrease in the coil interval of the stent. In perfusion cultures with stents, the proliferation of ECs was influenced by the local flow velocity distribution. When a stent with a CP value of 10 mm was used, the doubling time of ECs was 30.7 h, while the doubling time was 38.5 h when the CP was 5 mm. The doubling time of ECs was shorter at sites upstream of the stent wire where the velocity was higher than downstream of the wire. In conclusion, a single-coil stent can be used to modify hemodynamic factors, suggesting that improved stent design may facilitate rapid endothelialization after stent implantation.