A rat model of dual-flow liver machine perfusion system.

PURPOSE: As clinical liver perfusion systems use portal vein and artery flow, dual perfusion techniques are required even in small animal models in order to reproduce clinical setting. The aim of this study was to construct a new dual-flow perfusion system in rat model and optimized the oxygen supply to ensure the aerobic metabolization. METHODS: The dual-flow circuit was fabricated using rat liver and whole blood samples as perfusates. The oxygen supply was controlled according to the amount of dissolved oxygen in the perfusate. Perfusate parameters and adenosine triphosphate (ATP) levels were analyzed to evaluate organ function and metabolic energy state. Stored whole blood also tested the suitability as perfusate. RESULTS: Stored blood showed decrease oxygen delivery and liver function compared to fresh blood. Using fresh blood as perfusate with air only, the dissolved oxygen levels remained low and anaerobic metabolism increased. In contrast, with oxygen control at living body level, anaerobic metabolism was well suppressed, and tissue ATP content was increased. CONCLUSIONS: We developed a new dual-flow system that enable to reproduce the clinical settings. The perfusion system showed the possibility to improve the energy metabolic state of the perfused organ under appropriate partial pressure of oxygen.

Preliminary Observations of An Ex Vivo Normothermic Whole Blood Machine Perfusion in An Experimental Liver Transplant Porcine Model.

BACKGROUND: Even though transplantation is an essential treatment with no viable alternatives, a significant worldwide donor shortage persists. In this study, we assessed the metabolism of livers that underwent extended periods of circulatory death and subsequently conducted functional validation through transplantation to explore the feasibility of using livers from an uncontrolled donor after circulatory death (u-DCD). METHODS: A donor model simulating u-DCD was constructed using pigs. The prolonged warm ischemia time (WIT) was set to 60, 120, and 180 minutes, and the liver function was evaluated after 24 hours of perfusion using an originally developed normothermic perfusion system. Based on the results, functional confirmation by transplantation was performed on the 2 groups with prolonged WIT of 60 and 180 minutes. RESULTS: Based on the 24-hour perfusion of the liver alone, we evaluated the function by transplanting the WI 60-minute model and 180-minute model (N = 3 each). Warm ischemia was 73.5 ± 3.7 minutes and 188 ± 3 minutes in the 60-minute model and 180-minute model, respectively. In the model with 60 minutes of WI, one case survived until the endpoint, and 2 cases survived between 8 and 12 hours, whereas, in the model with 180 minutes of WI, they died within 6 hours. CONCLUSION: We constructed a completely uncontrolled circulatory arrest model without anticoagulation and showed the possibility of using u-DCD livers by ex vivo machine perfusion and transplantation.

A rat model of dual-flow liver machine perfusion system

Purpose: As clinical liver perfusion systems use portal vein and artery flow, dual perfusion techniques are required even in small animal models in order to reproduce clinical setting. The aim of this study was to construct a new dual-flow perfusion system in rat model and optimized the oxygen supply to ensure the aerobic metabolization. Methods: The dual-flow circuit was fabricated using rat liver and whole blood samples as perfusates. The oxygen supply was controlled according to the amount of dissolved oxygen in the perfusate. Perfusate parameters and adenosine triphosphate (ATP) levels were analyzed to evaluate organ function and metabolic energy state. Stored whole blood also tested the suitability as perfusate. Results: Stored blood showed decrease oxygen delivery and liver function compared to fresh blood. Using fresh blood as perfusate with air only, the dissolved oxygen levels remained low and anaerobic metabolism increased. In contrast, with oxygen control at living body level, anaerobic metabolism was well suppressed, and tissue ATP content was increased. Conclusions: We developed a new dual-flow system that enable to reproduce the clinical settings. The perfusion system showed the possibility to improve the energy metabolic state of the perfused organ under appropriate partial pressure of oxygen.

Rapid Metabolic Recovery of Donor Circulatory Death Liver Graft Using Whole Blood Perfusion: A Pig Study.

Ex vivo perfusion technology has been actively developed to solve the problem of severe donor shortage. In this study, the ex vivo metabolic characteristics of porcine donation after circulatory death (DCD) liver in short-term perfusion using whole or diluted blood were compared with those of the in vivo transplanted state to evaluate their initial response to resuscitation. METHODS: The porcine DCD model was constructed by clamping the thoracic aorta. After 60 min of blood flow cessation, retrieved livers were flushed with 500 mL of heparin saline (20 000 IU/L) followed by perfusion with 500 mL of cold histidine-tryptophan-ketoglutarate solution. The liver grafts were immersed in cold histidine-tryptophan-ketoglutarate solution for 60 min. Subsequently, normothermic ex vivo perfusion was performed with 20 000 IU/L of heparin added to the collected blood (whole blood group) or medium mixed with 10% whole blood (dilution group) for 3 h. Blood from the portal vein, the hepatic artery, and infra hepatica inferior vena cava was collected hourly and metabolomic analyses were performed. The other liver graft was heterotopically transplanted as a control (in vivo group). Each experiment was conducted once. RESULTS: The guanosine levels demonstrated similar fluctuating trends in the whole blood and in vivo groups. In contrast, the levels increased during the perfusion in the diluted blood group. Fluctuations in choline metabolism demonstrated similar trends in the whole blood and in vivo groups. CONCLUSIONS: Ex vivo machine perfusion with whole blood over a short time resulted in a metabolic trend similar to that in the in vivo model. Further studies in this regard are warranted to progress in the utilization of DCD organs.

DNA Origami Nanoplate-Based Emulsion with Nanopore Function.

Bio-inspired functional microcapsules have attracted increasing attention in many fields from physical/chemical science to artificial-cell engineering. Although particle-stabilised microcapsules are advantageous for their stability and functionalisation potential, versatile methods for their functionalisation are desired to expand their possibilities. This study reports a water-in-oil microdroplet stabilised with amphiphilic DNA origami nanoplates. By utilising DNA nanotechnology, DNA nanoplates were designed as a nanopore device for ion transportation and to stabilise the oil-water interface. Microscopic examination revealed the microcapsule formed by the accumulation of amphiphilic DNA nanoplates at the oil-water interface. Ion current measurements revealed the nanoplate pores functioned as channel to transport ions. These findings provide a general strategy for the programmable design of microcapsules to engineer artificial cells and molecular robots.

Nanopore Logic Operation with DNA to RNA Transcription in a Droplet System.

This paper describes an AND logic operation with amplification and transcription from DNA to RNA, using T7 RNA polymerase. All four operations, (0 0) to (1 1), with an enzyme reaction can be performed simultaneously, using four-droplet devices that are directly connected to a patch-clamp amplifier. The output RNA molecule is detected using a biological nanopore with single-molecule translocation. Channel current recordings can be obtained using the enzyme solution. The integration of DNA logic gates into electrochemical devices is necessary to obtain output information in a human-recognizable form. Our method will be useful for rapid and confined DNA computing applications, including the development of programmable diagnostic devices.

Amplification and Quantification of an Antisense Oligonucleotide from Target microRNA Using Programmable DNA and a Biological Nanopore.

This paper describes a strategy for autonomous diagnoses of cancers using microRNA (miRNA) and therapy for tumor cells by DNA computing techniques and nanopore measurement. Theranostics, which involves the combination of diagnosis and therapy, has emerged as an approach for personalized medicine or point-of-care cancer diagnostics. DNA computing will become a potent tool for theranostics because it functions completely autonomously without the need for external regulations. However, conventional theranostics using DNA computing involves a one-to-one reaction in which a single input molecule generates a single output molecule; the concentration of the antisense drug is insufficient for the therapy in this type of reaction. Herein we developed an amplification system involving an isothermal reaction in which a large amount of the antisense DNA drug was autonomously generated after detecting miRNA from small cell lung cancer. In addition, we successfully quantified the amount of the generated drug molecule by nanopore measurement with high accuracy, which was more accurate than conventional gel electrophoresis. This autonomous amplification strategy is a potent candidate for a broad range of theranostics using DNA computing.

Augmentation of the inhibitory effect of blue light on the growth of B16 melanoma cells by riboflavin.

We have demonstrated that blue light has anticancer effects in cultured cancer cells and tumor-bearing animals. Based on our experimental findings, in addition to cytostatic activity that suppresses the proliferation of B16 melanoma cells, blue light may exert cytocidal activity through interaction with vitamin(s) contained in the culture medium. The present study was undertaken to identify the specific vitamins with which blue light interacts and to investigate the factors responsible for its cytocidal activity. B16 melanoma cells were incubated in media supplemented with various vitamins and exposed to blue light for 10 min. Cell necrosis was observed only in media containing riboflavin (0.4 mg/l). The effects of other components of visible light on riboflavin were also studied. Riboflavin-containing media were exposed to light of each of the three primary colors (red, green and blue) and the effects on the colony-forming capacity of B16 melanoma cells were evaluated. Cell necrosis was induced only in media exposed to blue light. The effects of riboflavin increased in a concentration-dependent manner in the range from 0.3 to 1.0 mg/l in blue-light-exposed media and were antagonized by the presence of catalase (200 U/ml). These findings suggest that cell necrosis is probably induced by active oxygen species such as hydrogen peroxide formed by the reaction of riboflavin with blue light.

Blue light inhibits the growth of skin tumors in the v-Ha-ras transgenic mouse.

12-O-Tetradecanoylphorbol-13-acetate (TPA) was applied to the back skin of v-Ha-ras (TG-AC) female transgenic mice at a dose of 2.5 microg/200 microl twice a week for 9 weeks. The back skin was then exposed to blue light (wavelength, 470 nm; irradiance, 5.7 mW/cm2) for 1 h daily for 9 weeks. The mice to which TPA was applied developed skin tumors at 6 weeks after the start of application. The tumor incidence rates at 6, 7, 8 and 9 weeks after the start of application were 70%, 80%, 100% and 100%, respectively, and the numbers of tumors 1 mm or more in diameter were 1, 5, 10 and 19, respectively. In the mice that were exposed to blue light after TPA application, the tumor incidence rates were 10%, 40%, 60% and 80%, respectively, and the numbers of tumors 1 mm or more in diameter were 0, 2, 5 and 9, respectively. Histopathological examination of the skin revealed that TPA application induced diffuse hyperplasia, exaggerated keratinization, and papillomas in all 10 mice. A localized form of epidermal hyperplasia was also observed in 4 mice. The incidence rate of papillomas in the mice that were exposed to blue light after TPA application was lower and the degree of exaggerated keratinization was greater. Exaggerated keratinization was considered to represent a regressive change following exposure. These findings suggest that exposure to blue light may be a promising new approach in the treatment of skin tumors.

Inhibition of lung metastasis of B16 melanoma cells exposed to blue light in mice.

The effects of blue light on B16 melanoma cells and on the metastasis of these cells to the lungs were investigated in mice. The exposure of B16 melanoma cells to blue light in two 20-min sessions resulted in marked suppression of cell growth measured at 7 days after exposure. When these cells were harvested, re-inoculated into medium and incubated for a further 7 days, their growth activity returned to almost the same level as that of cultured cells from the non-exposure control group. The melanoma cells harvested after 7 days of incubation were injected intravenously into mice. In the non-exposure group, black nodules developed on the lung surface and the nodules increased in size over time. In the blue-light-exposure group, the development of such black nodules on the lung surface was delayed, and the nodules were smaller. Histopathological examination revealed that blue light suppressed the growth of metastatic tumor cells, and no increase in the number of melanin-containing cells or atypical cells was induced in the metastatic lesions. These results suggest that blue light suppresses the metastasis of B16 melanoma cells.

Effects of blue-light-exposure on growth of extracorporeally circulated leukemic cells in rats with leukemia induced by 1-ethyl-1-nitrosourea.

To explore the possibility of using blue light for extracorporeal circulation therapy in patients with leukemia, the effects of blue light on cell growth in vitro and in extracorporeally circulated blood of rats with leukemia were evaluated. When HL60 cells circulated extracorporeally using a peristaltic pump were exposed to blue light for 5 h, the growth of the cells was found to be markedly suppressed. Then, the blood of rats with erythroblastic leukemia, induced by the administration of tap water containing l-ethyl-l-nitrosourea (ENU) for 9-16 weeks, was circulated extracorporeally and exposed to blue light for 3 h. Lymphocytes were separated from the peripheral blood immediately after the end of blue-light-exposure and incubated for 7 days. The growth of leukemic cells was found to be significantly suppressed following exposure to blue light, whereas the growth of normal lymphocytes was unaffected. These findings suggest that cancer cells may be more sensitive to blue light than normal cells.

Blue light inhibits the growth of B16 melanoma cells.

Although a number of studies have been carried out to examine the biological effects of radiation and ultraviolet radiation (UV), little is known concerning the effects of visible light. In the present study, exposure of B16 melanoma cells to blue light (wavelength 470 nm, irradiance 5.7 mW / cm(2)) from a light-emitting diode (LED) inhibited cell growth in proportion to the period of exposure, with no increase observed in the number of dead cells. The number of B16 melanoma colonies that formed after exposure to blue light for 20 min was only slightly less than that in non-exposed controls, but the colony size as assessed by the area covered by colonies and cell counts per colony were markedly decreased. The percentages of G0 / G1 and G2 / M phase cells were markedly increased, with a reduction in S phase cells as determined by flow cytometry after exposure to blue light. Furthermore, analysis of the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA also showed a reduction in the percentage of S phase cells after exposure. These results indicate that blue light exerts cytostatic effects, but not a cytocidal action, on B16 melanoma cells.

Inhibition by long-term fermented miso of induction of gastric tumors by N-methyl-N'-nitro-N-nitrosoguanidine in CD (SD) rats.

The present study was designed to investigate the effects of fermented miso in the diet on the induction of gastric tumors by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in male CD (SD) rats. A total of 120 animals, 6 weeks of age, were divided into 6 groups and given MNNG (100 ppm) in the drinking water for 16 weeks. Starting 1 week before the carcinogen treatment the rats were fed a normal control MF solid diet, or the same diet containing 10% long-term fermented, medium- or short-term fermented miso, or 1% NaCl until the end of the MNNG exposure period. They were then maintained on the MF control diet and normal tap water until the autopsy time point at 52 weeks. The long-term fermented miso significantly reduced the size of the gastric tumors as compared with the other groups. The present results thus indicate that dietary supplementation with long-term fermented miso could act as a chemopreventive agent for gastric carcinogenesis.

Prevention by long-term fermented miso of induction of colonic aberrant crypt foci by azoxymethane in F344 rats.

The present study was designed to investigate the effects of fermented miso in the diet on the induction of aberrant crypt foci (ACF) by azoxymethane (AOM) in male F344 rats. A total of 50 rats, 8 weeks of age, were divided into 5 groups and given weekly subcutaneous injections of AOM (15 mg/kg body wt) for 3 weeks. Rats were fed a normal control MF solid diet, or solid diet containing 10% long-term fermented (aged), medium- or short-term fermented miso, or 2.2% NaCl for 5 weeks, starting one week before the first AOM dosing. It was found that, compared to the control (MF) diet, the long-term fermented diet significantly decreased (by 22.2%) ACF/colon, but increased (by 18.2%) the number of aberrant crypts (Acs)/focus. The latter was also increased by the medium-term fermented diet (by 25.3%). The PCNA labeling index was only affected by the short-term fermented diet (36.9% increase) and by 2.2% NaCl diet (27.2% increased). The present results indicate that aged or completely fermented miso supplemented into the diet, could act as a chemopreventive agent for colon carcinogenesis.