Cryopreservation of in vitro Shoot Tips of Ulluco (Ullucus tuberosus Cal.) Using D Cryo-Plate Method.

　　BACKGROUND: Maintenance of in vitro collections of ulluco (Ullucus tuberosus Cal.) is cumbersome and costly in an ex-situ genebank. An alternative method for long term preservation which is safe and cost-effective is required. OBJECTIVE: To apply a novel cryopreservation procedure using the cryo-plate system to improve the long-term conservation of ulluco. MATERIALS AND METHODS: Initially V and D cryo-plate methods were tested, subsequently the D cryo-plate method was selected for ulluco cryopreservation. The D cryo-plate procedures were optimized for post-LN regrowth procedures including cold-hardening, sucrose addition in alginate gel, and duration of LS treatment. Optimized procedures were tested with 11 ulluco lines. RESULTS: Shoot tips were isolated from cold-hardened shoots for 3-4 weeks at 5 degree C were excised to 1.0-1.5 mm long and 0.5 mm wide and precultured for 16h at 25 degree C on MS with 0.3 M sucrose. The shoot tips were attached on the cryo-plates by alginate gel with 0.4M sucrose. The cryo-plates with attached shoot tips were treated with 2.0 M glycerol and 1.0 M sucrose solution for 90 min at 25 degree C and dehydrated on filter paper in a Petri dish by air current flow at 25 degree C for 45 min before direct immersion in LN. This optimized procedure was applied to shoot tips of 11 ulluco lines, resulting regrowth ranging from 73 % to 97 %, with an average of 90 % post-LN regrowth. CONCLUSION: D cryo-plate is a practical and simple procedure for cryo-storage of in vitro grown ulluco shoot tips in an ex situ genebank.

Novel integrative methodology for engineering large liver tissue equivalents based on three-dimensional scaffold fabrication and cellular aggregate assembly.

A novel engineering methodology for organizing a large liver tissue equivalent was established by intergrating both 'top down' and 'bottom up' approaches. A three-dimensional (3D) scaffold was engineered comprising 43 culture chambers (volume: 11.63 cm(3)) assembled in a symmetrical pattern on 3 layers, a design which enables further scaling up of the device to a clinically significant size (volume: 500 cm(3)). In addition, an inter-connected flow channel network was designed and proved to homogenously deliver culture medium to each chamber with the same pressure drop. After fabrication using nylon-12 and a selective laser sintering process, co-cultured cellular aggregates of human hepatoma Hep G2 and TMNK-1 cells were loosely packed into the culture chambers with biodegradable poly-L-lactic acid fibre pieces for 9 days of perfusion culture. The device enabled increased hepatic function and well-maintained cell viability, demonstrating the importance of an independent medium flow supply for cell growth and function provided by the current 3D scaffold. This integrative methodology from the macro- to the micro-scale provides an efficient way of arranging engineered liver tissue with improved mass transfer, making it possible to further scale up to a construct with clinically relevant size while maintaining high per-volume-based physiological function in the near future.

ASSESSMENT OF MOLECULAR GENETIC STABILITY BETWEEN LONG-TERM CRYOPRESERVED AND TISSUE CULTURED WASABI (Wasabia japonica) PLANTS.

BACKGROUND: Maintaining the genetic integrity in long-term tissue cultured and cryopreserved plants is important for the conservation of plant genetic resources. OBJECTIVE: In this study, the genetic stability of cryopreserved wasabi shoot tips stored for 10 years at -150 degree C was visualized using Amplified Fragment Length Polymorphism (AFLP) and Methylation Sensitive Amplified Polymorphism (MSAP). MATERIALS AND METHODS: The study included plants derived from cryopreserved shoot tips after 10.5 years storage at -150 degree C (LN10yr), after 2 h storage at -196 degree C (LN2hr), cryopreservation controls (No LN cooling (TC)) and non-treated controls without LN cooling (LC). The donor plants for LN2hr, TC and LC were also maintained in vitro at 20 degree C for the same period. RESULTS: Neither technique detected genetic variations in either control or cryopreserved plants. Some mutations were noted in plants maintained in tissue culture for 10 years. Comparison of genome stability for TC and LN2hr plants showed only a minor change in DNA. However, when comparing the LC and Ln10yr, many differences were found. CONCLUSION: We conclude that cryopreservation is a superior conservation method compared to tissue culture in maintaining genetic stability for a long-term storage of wasabi germplasm.

Dehydration improves cryopreservation of mat rush (Juncus decipiens Nakai) basal stem buds on cryo-plates.

Two cryopreservation procedures using aluminium cryo-plates, termed V-Cryo-plate and D-Cryo-plate, were successfully developed for in vitro mat rush (Juncus decipiens Nakai) basal stem buds. Multiple stems induced in liquid MS medium containing 8.9 µM BA by roller culture were cut into small clumps, plated on solid MS medium and cultured for 1 week at 25 degree C. Clumps that had produced many buds were cold-hardened at 5 degree C for 1-2 months. The buds with basal stems were dissected from small clumps and precultured overnight at 25 degree C on solid MS medium containing 0.3 M sucrose. Precultured buds were placed on aluminium cryo-plates and embedded in calcium alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 25 degree C in loading solution (2 M glycerol + 1.0 M sucrose). In the D-Cryo-plate procedure, the buds were dehydrated to 27-25% moisture content (fresh weight) by placing the cryo-plates in the air current of a laminar flow cabinet for 2 to 3 h. In the V-Cryo-plate procedure, buds were dehydrated by immersing the cryo-plates in PVS2 vitrification solution for 40 min at 25 degree C. In both procedures, cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, cryo-plates were immersed in medium with 1.0 M sucrose for 20 min at room temperature. Regrowth of cryopreserved buds of line 'Kitakei 2' using D-Cryo-plate and V-Cryo-plate procedures, was 90% and 80%, respectively. The two procedures were applied to 20 additional mat rush lines. Using the V-Cryo-plate procedure resulted in regrowth ranging between 13.3 and 86.7%, with an average of 52.5%. The D-Cryo-plate led to regrowth ranging between 73.3 and 96.7%, with an average of 86.3%. The D-Cryo-plate procedure will facilitate cryostorage of mat rush germplasm.

V-cryo-plate procedure as an effective protocol for cryobanks: case study of mint cryopreservation.

A vitrification procedure using aluminium cryo-plates (V-Cryo-plate procedure) was successfully developed and adjusted for in vitro-grown mint (Mentha spp.) shoot tips. Shoots were cultured at 25°C on MS medium containing 0.088 M sucrose for 7 to 14 days after the last subculture. Shoot tips with a basal part (1-1.5 mm × 1 mm) were dissected from the shoots and precultured at 25°C for 1 day on the same medium. Precultured shoot tips were placed on aluminium cryo-plates with 10 wells and embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 25 degree C in 25 ml pipetting reservoirs filled with loading solution (2 M glycerol + 0.8 M sucrose). For dehydration, the cryo-plates were transferred and immersed in 25 ml pipetting reservoirs filled with PVS2 for 20 min at 25 degree C. Then the cryo-plates were transferred in uncapped 2 ml cryotubes and directly plunged into liquid nitrogen. For rewarming, shoot tips attached to the cryo-plates were immersed in cryotubes containing 2 ml 1 M sucrose solution at room temperature. Using this procedure, regrowth of cryopreserved shoot tips of line 'Fukuyamajisei' reached over 90 percent. This protocol was successfully applied to 16 additional Mentha lines, with regrowth ranging from 73 percent to 100 percent. This V-Cryo-plate method will facilitate the cryostorage of mint germplasm in our genebank.

Engineering of implantable liver tissues.

In this chapter, from the engineering point of view, we introduce the results from our group and related research on three typical configurations of engineered liver tissues; cell sheet-based tissues, sheet-like macroporous scaffold-based tissues, and tissues based on special scaffolds that comprise a flow channel network. The former two do not necessitate in vitro prevascularization and are thus promising in actual human clinical trials for liver diseases that can be recovered by relatively smaller tissue mass. The third approach can implant a much larger mass but is still not yet feasible. In all cases, oxygen supply is the key engineering factor. For the first configuration, direct oxygen supply using an oxygen-permeable polydimethylsiloxane membrane enables various liver cells to exhibit distinct behaviors, complete double layers of mature hepatocytes and fibroblasts, spontaneous thick tissue formation of hepatocarcinoma cells and fetal hepatocytes. Actual oxygen concentration at the cell level can be strictly controlled in this culture system. Using this property, we found that initially low then subsequently high oxygen concentrations were favorable to growth and maturation of fetal cells. For the second configuration, combination of poly-L: -lactic acid 3D scaffolds and appropriate growth factor cocktails provides a suitable microenvironment for the maturation of cells in vitro but the cell growth is limited to a certain distance from the inner surfaces of the macropores. However, implantation to the mesentery leaves of animals allows the cells again to proliferate and pack the remaining spaces of the macroporous structure, suggesting the high feasibility of 3D culture of hepatocyte progenitors for liver tissue-based therapies. For the third configuration, we proposed a design criterion concerning the dimensions of flow channels based on oxygen diffusion and consumption around the channel. Due to the current limitation in the resolution of 3D microfabrication processes, final cell densities were less than one-tenth of those of in vivo liver tissues; cells preferentially grew along the surfaces of the channels and this fact suggested the necessity of improved 3D fabrication technologies with higher resolution. In any case, suitable oxygen supply, meeting the cellular demand at physiological concentrations, was the most important factor that should be considered in engineering liver tissues. This enables cells to utilize aerobic respiration that produces almost 20 times more ATP from the same glucose consumption than anaerobic respiration (glycolysis). This also allows the cells to exhibit their maximum reorganization capability that cannot be observed in conventional anaerobic conditions.

Laser sintering fabrication of three-dimensional tissue engineering scaffolds with a flow channel network.

The fabrication of tissue engineering scaffolds for the reconstruction of highly oxygen-dependent inner organs is discussed. An additive manufacturing technology known as selective laser sintering was employed to fabricate a highly porous scaffold with an embedded flow channel network. A porogen leaching system was used to obtain high porosity. A prototype was developed using the biodegradable plastic polycaprolactone and sodium chloride as the porogen. A high porosity of 90% was successfully obtained. Micro x-ray CT observation was carried out to confirm that channels with a diameter of approximately 1 mm were generated without clogging. The amount of residual salt was 930 µg while the overall volume of the scaffold was 13 cm(3), and it was confirmed that the toxicity of the salt was negligible. The hydrophilization of the scaffold to improve cell adhesion on the scaffold is also discussed. Oxygen plasma ashing and hydrolysis with sodium hydroxide, typically employed to improve the hydrophilicity of plastic surfaces, were tested. The improvement of hydrophilicity was confirmed by an increase in water retention by the porous scaffold from 180% to 500%.

Efficacy of neutrophil elastase inhibitor on type A acute aortic dissection.

BACKGROUND: Surgery for type A acute aortic dissection (AAD) is associated with a high mortality and incidence of postoperative complications, including acute respiratory failure and coagulopathy. Aim of the study was to investigate the effects of sivelestat on pulmonary function and coagulopathy in patients undergoing surgery for AAD. METHODS: Sixty patients undergoing emergency ascending replacement for AAD were divided into two groups. Group I was administered sivelestat intravenously from the beginning of surgery until extubation. Group II was not treated with sivelestat. The platelet count, antithrombin III (AT III) level, leukocyte count, C-reactive protein (CRP) level, prothrombin time (PT), activated partial thrombin time (APTT), and prothrombin time-international normalized ratio (PT-INR) were measured. RESULTS: The postoperative decrease of AT III and the platelet count on admission to the intensive care unit (ICU) and 3 hours later were significantly less in group I. The leukocyte count and the values of CRP, PT, APTT, and PT-INR did not differ significantly between the groups. The duration of mechanical ventilation after surgery tended to be shorter in group I. CONCLUSIONS: Sivelestat significantly reduced the postoperative decreases in AT III and platelet count in patients undergoing emergency surgery for AAD.

Pulmonary embolism with myomatous erythrocytosis syndrome and extreme obesity.

We here report a case of a 53-year-old woman requiring pulmonary embolectomy for acute massive pulmonary embolism caused by a huge uterine myoma compressing veins in the pelvis and extreme obesity. She was also diagnosed as having myomatous erythrocytosis syndrome, a rare disease associated with secondary polycythemia. The polycythemia improved after a hysterectomy which was performed after pulmonary embolectomy.

Proton pump inhibitors may increase the risk of delayed bleeding complications after open heart surgery if used concomitantly with warfarin.

OBJECTIVES: The American Food and Drug Administration has suggested that proton pump inhibitors increase the international normalized ratio (INR) when used concomitantly with warfarin, by being metabolized by cytochrome P450 2C19. We therefore reviewed patients taking warfarin. METHODS AND RESULTS: Two hundred and forty patients who took warfarin after surgery were divided into two groups: Group I (n = 114) had rabeprazole (10 mg/day) and Group II (n = 126) had lansoprazole (15 mg/day). The initial dose of warfarin was 3 mg and INR was initially assessed on postoperative day 4. Initial INR was significantly lower in Group I (1.66 +/- 0.87) than in Group II (2.06 +/- 1.03, P = 0.0011). Delayed cardiac tamponade and hemothorax occurred as complications in 6 and 1 patients, respectively, in Group II from 5 days to 3 months postoperatively. At the time of the occurrence of complications, the average INR increased to 3.95 (range from 3.11 to 5.86). There were no patients with delayed bleeding in Group I ( P = 0.015). CONCLUSIONS: These results suggest that lansoprazole emphasizes the effects of warfarin. Rabeprazole could be safely used concomitantly with warfarin.

[Reoperation for a Starr-Edwards ball valve prosthesis implanted in mitral position 31 years ago].

The patient is a 61-year-old woman who had undergone mitral valve replacement with the Starr-Edwards cloth-covered ball valve 31 years ago. She had dyspnea on effort 1 month before admission. The echocardiography revealed mitral and tricuspid regurgitation. Re-replacement of the mitral prosthetic valve with an ATS valve and tricuspid annuloplasty were successfully performed without any complication. The cloth wear of the Starr-Edwards ball valve cage was recognized and no thrombus was found at operation. To our knowledge, there has been no such a reoperative case who underwent so long postoperative period after initial implantation of the Starr-Edwards ball valve in Japan. This experience made us realize again the importance of attention to the complications related to a prosthetic valve like a cloth wear during long-term follow-up for the Starr-Edwards ball valve.

Three-dimensional STEM for observing nanostructures.

A new scanning transmission electron microscope has been developed for three-dimensional (3D) observations of nanostructures. Using double spherical fulcra, accurate eucentric rotation was achieved. Cylindrical specimens for 3D-observation were prepared by a microsampling technique using a focused ion beam. Copper via-holes of a semiconductor memory device and ZnO particles were observed by the 3D-STEM from different directions, and 3D-data of the ZnO particles were successfully reconstructed in a topography mode.

Pathological findings of tissue reactivity of gelatin resorcin formalin glue: an autopsy case report of the repair of ventricular septal perforation.

A seventy-three-year-old man was treated for ventricular septal perforation with Gelatin Resorcin Formalin (GRF) glue. The patient died of multiple organ failure 36 days after the surgery. In autopsy, macroscopically, the inferior wall was reconstructed successfully by the GRF glue. Furthermore, microscopic study revealed the excellent growth of collagen and elastic fiber where the GRF was glued. No infiltration of inflammatory cells was evident. There have been no reports that the safety and efficacy of GRF glue was pathologically proven in an autopsy case.

Removal of a chronically infected bipolar pacemaker electrode: a successful case report.

A chronic infected bipolar pacemaker electrode with a fin tip was successfully removed 7 years and 9 months after its original implantation from a 72-year-old Japanese man, using the Cook pacemaker lead extraction system. The locking stylet could not advance to the lead tip over the positive pole because of firm adhesions. Because the scar tissue between the positive pole and myocardium could not be freed by the inner sheath, it was disrupted by the slanted end of the outer sheath. The firmest adhesion was on the positive pole, not on the fin tip. The complete extraction success rate of bipolar tined or fin leads is worse than for other types of leads. When extracting a bipolar pacemaker lead, dissection of the positive pole from scar tissue should be taken into account in addition to the lead tip. Rotating the slanted end of the outer sheath is a useful technique when dissecting firm adhesions.

Cryopreservation of lateral buds of in vitro-grown innala plants (Solemostemon rotundifolius) by vitrification.

We succeeded in cryopreserving of innala (Solenostemon rotundifolius) in vitro-grown young lateral buds by vitrification. Nodal segments from in vitro-grown shoots (2-4 mm in length) were cultured on MS medium containing 0.1M sucrose in Petri dishes for 3 weeks under 16-h photoperiod at 25 degree C. This pre-growth induced a large number of uniform young lateral buds. Nodal segments (0.5 to 1.0 mm in length) with two lateral buds were dissected from the shoots and precultured with 0.3 M sucrose for 2 days at 25 degree C. They were then treated with loading solution containing 2 M glycerol and 0.4 M sucrose (LS solution) for 20 min at 25 degree C and dehydrated with the PVS2 vitrification solution for 18 min at 25(C prior to either rapid immersion in liquid nitrogen. Surviving lateral buds resumed growth within 3 days and developed shoots without intermediary callus formation. The average growth recovery after cryopreservation amounted to 85%.

Newly developed encapsulation-dehydration protocol for plantcryopreservation.

A simplified and efficient encapsulation-dehydration protocol, which is a compromise between vitrification and encapsulation-dehydration, was presented for plant cryopreservation. In this protocol, during the encapsulation process, the apices precultured with 0.3 M sucrose for 16 h were simultaneously osmoprotected with a mixture of 02 M glycerol plus 0.4 M sucrose for 1 h. These encapsulated apices were directly dehydrated with dry silica gel prior to a plunge into LN without the pretreatment of 0.8M sucrose for 16 h. This protocol produced much higher rates of recovery growth in the three plant species tested (wasabi, chrysanthemum, and mint) than those cryopreserved by the conventional encapsulation-dehydration. This protocol also considerably reduced the time needed for the cryogenic procedure. Thus, this new protocol appears promising for cryopreservation of shoot apices and other explants.

Doppler features of occlusion of the internal thoracic artery due to preoperative branch embolization.

A 61-year-old male was referred to the surgical ward by cardiologists because of a diagnosis of unstable angina with 3-vessel disease. On preoperative left internal thoracic arteriography, a large first intercostal branch was found at the proximal portion. Selective arterial embolization of the branch of the left internal thoracic artery (LITA) was carried out preoperatively. At 2 days after embolization, the Doppler peak flow velocity and diameter of the LITA were increased and enlarged compared with before the procedure. However, a large reverse wave following after the first systolic peak flow of the LITA was newly detected after embolization. Upon operation, the LITA was found to be occluded at the 2nd intercostal space due to thrombus formation. Therefore, the right internal thoracic artery was anastomosed to the left anterior descending artery and coronary reversed saphenous vein grafts were joined to segment 4PD of the right coronary artery. The postoperative course was uneventful. There has been no previous report of an LITA branch being embolized preoperatively. It was possible to diagnose the graft problem by detecting the altered Doppler wave form of the LITA.

[An objective measurement of softening of uterine cervix using tactile sensor for hardness].

The authors studied an objective measurement for cervical softening by means of a tactile sensor equipped with a piezoelectric swing element made from ceramic. The hardness of objects was indicated by changes in frequency (delta f) measured by this sensor. The authors measured the hardness of cervixes in 66 (non pregnant, pregnant, delivering and postpartum) Sprague-Dawley rats with the sensor. After the measurement of delta f, the rats' cervixes were removed, and the ratios of their dry weight to wet weight (weight-ratio) and hydroxyproline contents were measured. The delta f of rats' cervixes increased during pregnancy and became maximum at delivery. The weight-ratio also increased and correlated with delta f (r = 0.812). The hydroxyproline content decreased during pregnancy and correlated with delta f (r = 0.674). The hardness of the cervixes of 26 non pregnant and 192 pregnant women and their Bishop scores were also measured. The delta f of pregnant women's cervixes increased significantly in comparison with those of non pregnant women. The delta f of cervixes in primiparas correlated with the progress of pregnancy (r = 0.408). A moderate correlation between the delta f of cervixes and Bishop scores was also observed in the pregnant women. These results show that the tactile sensor may be able to be used in the objective measurement of hardness of the uterine cervix.