ABSTRACT
Grafts from donors after cardiac death (DCD) have greatly contributed to expanding the donor organ pool. This study aimed to determine the benefits of subnormothermic extracorporeal membrane oxygenation (ECMO) and hypothermic machine perfusion (HMP) in a porcine model of DCD liver. Female domestic crossbred Large Yorkshire and Landrace pigs weighing approximately 20 kg were used. The abdominal aorta and inferior vena cava were cannulated and connected to an ECMO circuit for in situ perfusion of the abdominal organs at 22 °C for 60 min, 45 min after cardiac death. The pigs were divided into the cold storage (CS) group (n = 3), where liver grafts were preserved at 4 °C, and the HMP group (n = 3), where liver grafts were preserved by HMP at 8-10 °C. After 4 h of preservation, liver function was evaluated using an isolated liver reperfusion model for 2 h. Although the difference was insignificant, the liver effluent enzyme levels in the HMP group were lower than those in the CS group. Furthermore, morphological findings showed fewer injured hepatocytes in the HMP group than in the CS group. The combined use of in situ subnormothermic ECMO and HMP was beneficial for the functional improvement of DCD liver grafts.
ABSTRACT
Midthermic machine perfusion (MMP) of post-circulatory arrest donor liver grafts has the advantage of preserving the functional ultrastructure of hepatocytes in donor grafts. It was reported that oxygenation during MMP reduces portal venous resistance and increases bile production. The MMP with hemoglobin-based oxygen vesicles (HbV) keeps the lower aspartate aminotransferase level (an indicator of liver injury) and maintains the functional ultrastructure of mitochondria in the hepatocytes. To evaluated differences of ultrastructural damages in donor livers between the MMP with and without HbV, porcine liver grafts after 60 min of warm ischemia were perfused at 22°C for 4 h with or without HbV, and a part of liver grafts were analyzed by transmission electron microscopy (TEM) and osmium-maceration scanning electron microscopy (OM-SEM). The remaining grafts were perfused with autologous blood at 38°C for 2 h in an isolated liver reperfusion model (IRM) that mimics the inside of the body after transplantation, and then analyzed by TEM and OM-SEM. Hepatocytes after MMP had small round mitochondria with rod-shaped cristae and reticulovesicular rough endoplasmic reticulum (rER) in both HbV(+) and HbV(-) livers. After IRM of HbV(+) livers, the well-developed lamellar rER was often found in hepatocytes. Liver sinusoidal endothelial cells (LSECs) after MMP contained some large vacuolar structures containing amorphous garbage in the cytoplasm, and their size along with appearance frequency were smaller and lower, respectively, in HbV(+) livers than HbV(-). Oxygenation during the MMP by using HbV suppressed the ultrastructural damages in donor livers, in particular for the LSECs. RESEARCH HIGHLIGHTS: Liver sinusoidal endothelial cells after midthermic machine perfusion had large vacuolar organelles with amorphous garbage. Oxygenation during the perfusion made them less and smaller, ultrastructurally supporting its utility.
Subject(s)
Liver Transplantation , Swine , Animals , Humans , Oxygen , Endothelial Cells , Organ Preservation , Perfusion , Living Donors , Liver/ultrastructure , Death , HemoglobinsABSTRACT
BACKGROUND AND OBJECTIVE: In recellularization, cell-seeding efficiency refers to the uniform distribution of cells across the decellularized organ, which should be enhanced to ensure effective functioning. During cell seeding, flow dynamics influence the distribution of cells because the driving force of cell movement is the fluid force. However, after decellularization, because of flow permeability through the vessel wall, the fluid pressure and velocity in the vessels of vascular trees are significantly reduced compared with those in the native organ, which might affect cell seeding efficiency. Therefore, it is necessary to assess the flow characteristics in the vessels of decellularized organs to select appropriate seeding conditions. Although electrical analog models have been widely used to investigate the flow distribution in organs, current models do not reflect the permeable conditions. This study proposes a model to extend the conventional electrical analog model to simulate the flow characteristics in decellularized organs. METHODS: A resistor reflecting permeable flow was added to the original electrical analog model to describe the permeable conditions in the decellularized organs. Decellularization and pressure drop measurements of the kidney were also conducted for model development, calibration, and validation. The developed model was then applied to a decellularized kidney to reveal changes in flow characteristics. RESULTS: The resistance calculation of permeable flow was determined for each generation of vascular trees. The coefficient of permeability can be indicated by the measured flow exiting through the outlet or the pressure drop across the decellularized organ. The developed permeability model had a qualitative and quantitative agreement with the experimental data without calibration. The results of the permeability model for the decellularized kidney indicated significant reductions of up to 70% in the flow rate and pressure of the organ compared to the native kidney. CONCLUSIONS: The developed model can simulate the flow characteristics in each individual vessel of decellularized organs. The results from the model can be used to assess the optimal flow rate condition for the cell seeding process.
Subject(s)
Hydrodynamics , Tissue ScaffoldsABSTRACT
BACKGROUND: The use of grafts from donors after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. This study aims to determine the benefits of extracorporeal membrane oxygenation (ECMO) and hypothermic oxygenated machine perfusion (HOPE) in a large animal model of DCD liver. METHODS: After cardiac arrest, the abdominal aorta and the inferior vena cava were cannulated and connected to an ECMO circuit. Porcine livers were perfused in situ with ECMO at 22°C for 60 minutes after 45 minutes of cardiac death. Then, the livers were perfused for 4 hours by cold storage (CS) or HOPE. In group 1, non-in situ ECMO and grafts were preserved by HOPE. In group 2, in situ ECMO and grafts were preserved by HOPE. In group 3, in situ ECMO and grafts were preserved by CS. After preservation, all grafts were evaluated using an isolated reperfusion model (IRM) with autologous blood for 2 hours. RESULTS: During HOPE, aspartate aminotransferase (AST) levels and hepatic arterial pressure in group 2 tended to be lower than in group 1. Hematoxylin-eosin staining findings after HOPE showed more massive sinusoidal congestion and hepatocyte cytoplasmic vacuolization in group 1 than in group 2. The AST and LDH levels in group 2 at the start-up of IRM tended to be lower than in group 1. CONCLUSIONS: The combined use of in situ subnormothermic ECMO and HOPE is essential for the functional recovery of DCD liver grafts.
Subject(s)
Liver Transplantation , Organ Preservation , Swine , Animals , Liver/surgery , Perfusion , DeathABSTRACT
To ensure the functional properties of an organ generated by the process of decellularization and recellularization, the initial density and distribution of seeding cells in the parenchymal space should be maximized. However, achieving a uniform distribution of cells across the entire organ is not straightforward because of vessel occlusion. This study assessed vessel occlusion during recellularization under different conditions. A combination of the electrical analog permeability (EPA) model, computational fluid dynamics (CFD), and discrete element method (DEM) was employed to describe the vessel occlusion phenomenon. In particular, realistic flow distributions in vascular trees of the decellularized organ were indicated by the EPA model. The cell suspension flow was modeled by a coupled CFD-DEM model, whereby living cells were presented as a discrete phase (solved by the DEM solver), and the culture medium was modeled as the fluid phase (solved by CFD solver). The cell suspension velocity was reduced up to 47% after decellularization, which directly affected cell movement. Simulation results also indicate that the occurrence of vessel occlusion was promoted by gravity direction in the asymmetric bifurcation and increased as the cell concentration increased. The assessment of vessel occlusion under different conditions was quantitatively investigated. The model provides insights into the dynamics of cells in the vessel compartment, allowing for the selection of optimum seeding parameters for the recellularization process.
Subject(s)
Computer Simulation , Tissue Culture Techniques , Vascular DiseasesABSTRACT
BACKGROUND AND AIM: There are very few reports comparing the use of the University of Wisconsin solution and histidine-tryptophan-ketoglutarate solution as machine perfusion solutions for marginal liver grafts. We aimed to clarify whether the use of the histidine-tryptophan-ketoglutarate solution in hypothermic machine perfusion improves the split-liver graft function in a large animal model. METHODS: Porcine split-liver grafts were created by 75% liver resection. Hypothermic machine perfusion experimental groups were divided as follows: Group 1, perfusate, University of Wisconsin gluconate solution (UW group; n = 5), and Group 2, perfusate, histidine-tryptophan-ketoglutarate solution (HTK group; n = 4). After 4 h of preservation, the liver function was evaluated using an isolated liver reperfusion model for 2 h. RESULTS: In the HTK group, the portal vein and hepatic artery resistance during hypothermic machine perfusion and the portal vein resistance during isolated liver reperfusion were lower than those in the UW group. In addition, the total Suzuki score for hepatic ischemia-reperfusion injury in the HTK group was significantly better than that in the UW group. The number of anti-ETS-related genes staining-positive sinusoid epithelial cell nuclei in the HTK group was higher than that in the UW group (not significant). CONCLUSIONS: The histidine-tryptophan-ketoglutarate solution can be perfused with lower vascular resistance than the University of Wisconsin solution, reducing shear stress and preventing sinusoid epithelial cell injury in marginal grafts used as split-liver grafts.
Subject(s)
Organ Preservation Solutions , Organ Preservation , Animals , Swine , Organ Preservation Solutions/pharmacology , Liver , Glutathione/pharmacology , Insulin , PerfusionABSTRACT
BACKGROUND: The machine perfusion (MP) preservation including hypothermic MP (HMP) and midthermic MP (MMP) has been considered as a promising strategy to preserve the functions of liver donated after cardiac death. The importance of understanding liver sinusoidal endothelial cells (LSEC) damage in regulating liver injury during MP has been emphasized. However, the ultrastructural changes in the LSEC and sinusoids around them after MP are unclear. AIM: To investigate the ultrastructural changes in the LSEC and sinusoids around them after MP. METHODS: Porcine liver grafts undergo a warm ischemia time of 60 minutes perfused for 4 h with modified University of Wisconsin gluconate solution. Group A grafts were preserved with HMP at 8 °C constantly for 4 h. Group B grafts were preserved with a rewarming solution at 22 °C by MMP for 4 h. Then the ultrastructural changes in the LSEC and sinusoids in Group A and B were comparatively analyzed by using osmium-maceration scanning electron microscopy with complementary transmission electron microscopy methods. RESULTS: An analysis of the LSEC after warm ischemia revealed that mitochondria with condensed-shaped cristae, abnormal vesicles, reduction of ribosomes and the endoplasmic reticulum (ER) surround the mitochondria appeared. The MP subsequent after warm ischemia alleviate the abnormal vesicles and reduction of ribosomes in LSEC, which indicated the reduction of the ER damage. However, MMP could restore the tubular mitochondrial cristae, while after HMP the condensed and narrow mitochondrial cristae remained. In addition, the volume of the sinusoidal space in the liver grafts after MMP were restored, which indicated a lower risk of pressure injury than HMP. CONCLUSION: MMP alleviates the ER damage of LSEC by warm ischemia, additionally restore the metabolism of LSEC via the normalization of mitochondria and prevent the share stress damage of liver grafts.
Subject(s)
Organ Preservation Solutions , Organ Preservation , Animals , Humans , Death , Endothelial Cells , Liver/metabolism , Organ Preservation/methods , Organ Preservation Solutions/pharmacology , Perfusion/methods , SwineABSTRACT
Transmembrane ion transport under tonicity imbalance has been investigated using a combination of low frequency-electrical impedance spectroscopy (LF-EIS) and improved ion transport model, by considering the cell diameterd[m] and the initial intracellular ion concentrationcin[mM] as a function of tonicity expressed by sucrose concentrationcs[mM]. The transmembrane ion transport is influenced by extracellular tonicity conditions, leading to a facilitation/inhibition of ion passage through the cell membrane. The transmembrane transport coefficientP[m s-1], which represents the ability of transmembrane ion transport, is calculated by the extracellular ion concentrations obtained by improved ion transport model and LF-EIS measurement.Pis calculated as 4.11 × 10-6and 3.44 × 10-6m s-1atcsof 10 and 30 mM representing hypotonic condition, 2.44 × 10-6m s-1atcsof 50 mM representing isotonic condition, and 3.68 × 10-6, 5.16 × 10-6, 9.51 × 10-6, and 14.89 × 10-6m s-1atcsof 75, 100, 125 and 150 mM representing hypertonic condition. The LF-EIS results indicate that the transmembrane ion transport is promoted under hypertonic and hypotonic conditions compared to isotonic condition. To verify the LF-EIS results, fluorescence intensityF[-] of extracellular potassium ions is observed to obtain the temporal distribution of average potassium ion concentration within the region of 3.6µm from cell membrane interfacecROI[mM]. The slopes of ∆cROI/cROI1to timetare 0.0003, 0.0002, and 0.0006 under hypotonic, isotonic, and hypertonic conditions, wherecROI1denotes initialcROI, which shows the same tendency with LF-EIS result that is verified by the potassium ion fluorescence observation.
Subject(s)
Dielectric Spectroscopy , Potassium , Ion Transport , Ions , Osmolar Concentration , Potassium/metabolismABSTRACT
Two equations have been developed from multi-frequency measurements of blood impedance Zb for a simultaneous electrical online estimation of changes in blood hematocrit ΔH [%] and temperatures ΔT [K] in cardiopulmonary bypass (CPB). Zb of fixed blood volumes at varying H and T were measured by an impedance analyzer and changes in blood conductivity σb and relative permittivity εb computed. Correlation analysis were based on changes in σb with H or T at f = 1 MHz while H and T equations were developed by correlating changes in εb with H and T at dual frequencies of f = 1 MHz and f = 10 MHz which best capture blood plasma Zp and red blood cell cytoplasm Zcyt impedances respectively. Results show high correlations between σb and H (R2 = 0.987) or σb and T (R2 = 0.9959) indicating dependence of the electrical parameters of blood on its H and T. Based on computed εb, changes in blood hematocrit ΔH and temperature ΔT at a given time t are estimated as ΔH(t) = 1.7298Δεb (f = 1 MHz) - 1.0669Δεb (f = 10 MHz) and ΔT(t) = -2.186Δεb (f = 1 MHz) + 2.13Δεb (f = 10 MHz). When applied to a CPB during a canine mitral valve plasty, ΔH and ΔT had correlations of R2 = 0.9992 and R2 = 0.966 against H and T respectively as measured by conventional devices.
Subject(s)
Cardiopulmonary Bypass , Animals , Dogs , Cardiopulmonary Bypass/methods , Hematocrit , Temperature , Electric ImpedanceABSTRACT
This study examined the efficacy of end-ischemic hypothermic oxygenated machine perfusion preservation (HOPE) using an originally developed machine perfusion system for split-liver transplantation. Porcine split-liver grafts were created via 75% liver resection after 10 min of warm ischemia. In Group 1, grafts were preserved by simple cold storage (CS) for 8 h (CS group; n = 4). In Group 2, grafts were preserved by simple CS for 6 h and end-ischemic HOPE for 2 h (HOPE group; n = 5). All grafts were evaluated using an isolated ex vivo reperfusion model with autologous blood for 2 h. Biochemical markers (aspartate aminotransferase and lactate dehydrogenase levels) were significantly better immediately after reperfusion in the HOPE group than in the CS group. Furthermore, the HOPE group had a better histological score. The levels of inflammatory cytokines (tumor necrosis factor-α, interferon-γ, interleukin-1ß, and interleukin-10) were significantly lower after reperfusion in the HOPE group. Therefore, we concluded that end-ischemic HOPE for split-liver transplantation can aid in recovering the graft function and reducing ischemia-reperfusion injury. HOPE, using our originally developed machine perfusion system, is safe and can improve graft function while attenuating liver injury due to preservation.
Subject(s)
Cold Ischemia , Liver Transplantation/methods , Organ Preservation/methods , Oxygen/pharmacology , Warm Ischemia , Animals , Female , Hepatocytes/metabolism , Inflammation/metabolism , Liver/pathology , Organ Preservation Solutions/pharmacology , Perfusion , Reactive Oxygen Species , Reperfusion , Reperfusion Injury/pathology , SwineABSTRACT
The seeding of cells into an organ is an important step in cell therapy because the final functional properties of the organ are related to the initial cell distribution throughout the organ. However, vessel occlusion is a serious problem that prevents uniform distribution of the cells in the entire organ. Understanding the mechanism of vessel occlusion can help optimize the seeding process. In this study, the vessel occlusion phenomenon under perfusion conditions during cell seeding was investigated. First, we applied a microfluidic system that enabled the observation of the occlusion events during injection. Second, we applied a multiphase numerical model that can describe the cell-cell interactions and cell-fluid interactions to investigate the vessel occlusion phenomenon during the seeding process. In particular, the effects of cell concentration and flow rate were investigated. The results indicate the importance of cell-cell interactions and cell-vessel interactions for the occurrence of vessel occlusion. In addition, it is found that the probability of occurrence of vessel occlusion increases with the increase in cell concentration and decrease in flow rate. The simulation model can help determine the optimum parameters to enhance cell seeding efficiency.
Subject(s)
Blood Vessels/pathology , Cell Culture Techniques , Computer Simulation , Models, Biological , Permeability , RheologyABSTRACT
Low-frequency impedance-based (LFI) cell discrimination as a novel non-destructive and non-invasive cell discrimination is proposed. LFI cell discrimination discriminates the cell type by considering an ion transport model in cell suspension. Ion transport model in cell suspension is constructed on the basis of Fick's laws of diffusion in the extracellular region under ion permeability P which represents the characteristics of cell type. P is achieved using the ion transport model equation through an iterative curve fitting to an ion concentration in extracellular region obtained from low-frequency impedance which is assumed to be linearly related to the ion concentration in extracellular region. In experiment, the electrical impedance spectra from the frequency of 200 kHz to 2.0 MHz are measured over time during producing ions from intracellular region to extracellular one in cell suspension using an impedance analyzer and an interdigitated array electrode system. As a target cell type, two different cell types based on Medical Research Council 5 (MRC-5), which are different in intracellular component are used. The curve fitting is performed for the low-frequency impedance at 200 kHz at which impedance reflects the ion concentration in extracellular region in order to obtain P of each cell type. As a result, each cell type has its own P. The proposed LFI cell discrimination successfully discriminates the cell type.
Subject(s)
Electric Impedance , Electrodes , Ion TransportABSTRACT
The effects of each type of machine perfusion preservation (MP) of liver grafts donated after cardiac death on the bile canaliculi of hepatocytes remain unclear. We analyzed the intracellular three-dimensional ultrastructure of the bile canaliculi and hepatocyte endomembrane systems in porcine liver grafts after warm ischemia followed by successive MP with modified University of Wisconsin gluconate solution. Transmission and osmium-maceration scanning electron microscopy revealed that lumen volume of the bile canaliculi decreased after warm ischemia. In liver grafts preserved by hypothermic MP condition, bile canaliculi tended to recover in terms of lumen volume, while their microvilli regressed. In contrast, midthermic MP condition preserved the functional form of the microvilli of the bile canaliculi. Machine perfusion preservation potentially restored the bile canaliculus lumen and alleviated the cessation of cellular endocrine processes due to warm ischemia. In addition, midthermic MP condition prevented the retraction of the microvilli of bile canaliculi, suggesting further mitigation of the damage of the bile canaliculi.
Subject(s)
Bile Canaliculi/ultrastructure , Death , Liver/ultrastructure , Organ Preservation , Animals , Female , Hepatocytes/ultrastructure , Liver Transplantation , Perfusion , Swine , Temperature , Warm IschemiaABSTRACT
BACKGROUND: The gallbladder filling and emptying cycle controls bile flow into the duodenum. Disruption of this emptying and refilling cycle leads to bile stasis and subsequent gallstone formation. The bile flow rate is inversely proportional to its viscosity. Moreover, bile is a complex material with varying density in different people because of its diverse components. These density changes alter the viscosity. Prediction of shear viscosity from density measurements may thus contribute to medical assessment and treatment of the patient. OBJECTIVE: To investigate the relationship between the shear viscosity and density of bile. METHODS: Natural and filtered bile samples were prepared for density, viscosity and pressure drop measurements. The density and shear viscosity were measured by a density meter and viscometer, respectively, and a relationship was established between them. Measurements of the pressure drop in a glass tube were used to estimate the apparent viscosity and compared with shear viscosity obtained with the viscometer. RESULTS: The natural bile with higher density showed a greater shear viscosity than the filtered bile, especially at low shear rates. Bile viscosity was proportional to density. In addition, the pressure drop of the natural samples was greater than that of the filtered sample. The viscosity measured by the viscometer deviated from the results obtained using Poiseuille's law and the tube pressure drop measurements. The density difference between the natural and filtered bile samples was smaller than 1%. However, the viscosities of the natural and filtered bile samples varied by up to >30%. CONCLUSIONS: Porcine bile viscosity is proportional to density. Based on the bile density, one can easily evaluate the shear viscosity using a linear relationship. The density considerably influences the bile viscosity, with small density increases greatly increasing the shear viscosity.
Subject(s)
Bile , Blood Viscosity , Gallstones , Animals , Humans , Swine , ViscosityABSTRACT
BACKGROUND Split-liver transplantation can be useful in situations of limited donor resources. However, novel preservation methods are required to help the recipient recover from severe ischemic reperfusion injury incurred due to receiving a relatively small liver graft. MATERIAL AND METHODS Our experiment was performed using porcine livers without warm ischemia time, assuming a brain-dead organ. We made porcine split-liver grafts by 75% liver resection at the back table and divided the specimens into 4 groups. Group 1 was preserved with simple cold storage after splitting (CS; n=3), Group 2 was preserved with hypothermic perfusion preservation (HMP) after splitting (SBP; n=3), Group 3 was preserved with HMP after splitting under perfusion preservation (SDP; n=4), and Group 4 had the whole liver perfused as control grafts (Whole Liver; n=3). To assess potential methods of preservation and their effects, all grafts were evaluated by an ex vivo isolated liver reperfusion model using diluted autologous blood. RESULTS Portal vein pressure resistances during reperfusion were low in Group3 (SDP). Hepatic artery pressure resistances during reperfusion were markedly higher in Group 1(CS) than in the other groups. The levels of AST and LDH were high and increased at 2 h after reperfusion in Group 1 (CS). The histological findings show that the liver cell structure was irregular in Group 1 (CS) but remained regular in Groups 2 (SBP) and 3 (SDP). Histological Suzuki scores were also significantly better in Groups 2 (SBP) and 3 (SDP) compared with Group 1 (CS). CONCLUSIONS Splitting the liver under machine perfusion preservation may help restore the function and reduce ischemia-reperfusion injury.
Subject(s)
Liver Transplantation/methods , Liver/surgery , Animals , Liver/blood supply , Models, Animal , Organ Preservation/methods , Perfusion/methods , Swine , Warm IschemiaABSTRACT
BACKGROUND: The present study examined the impact of oxygenated machine perfusion on preservation of liver grafts donated after cardiac death by measuring sinusoidal endothelial injury and microcirculatory disturbances. MATERIALS AND METHODS: Fifteen porcine livers were retrieved 60 min after warm ischemia and allocated into three groups as follows: (1) CS group: static cold storage, (2) HMP group: oxygenated hypothermic perfusion preservation, (3) SNMP group: oxygenated subnormothermic perfusion preservation. The liver grafts donated after cardiac death were preserved for 4 h in different treatment conditions mentioned previously, then subject to ex vivo reperfusion for 2 h using diluted allogeneic blood. The hemodynamic parameters, liver function tests, tissue adenosine triphosphate (ATP) levels, and immunohistochemical findings were investigated. RESULTS: The number of sinusoidal epithelial cells and trabecular structures were maintained after 4 h of preservation in the CS, HMP, and SNMP group. Liver tissue ATP levels after 4 h of preservation in the HMP and SNMP groups were significantly higher compared with that in the CS group. The sinusoidal epithelial cells were significantly exfoliated to a more severe extent in the CS group than in the HMP and SNMP groups. Intrasinusoidal platelet aggregation occurred more frequently in the CS group than in the HMP and SNMP groups. CONCLUSIONS: The results indicated that oxygenated machine perfusion preservation was important to prevent the depletion of tissue ATP and maintain sinusoidal homeostasis regardless of the perfusate temperature. Our findings suggest oxygenated machine perfusion preservation as an effective alternative to static cold storage.
Subject(s)
Liver Transplantation , Liver/blood supply , Microcirculation , Organ Preservation , Perfusion/methods , Animals , Endothelium, Vascular/pathology , Liver/metabolism , Liver/pathology , Oxygen Consumption , SwineABSTRACT
Improvement of machine perfusion (MP) technologies is required to enhance organ quality for donor after cardiac death (DCD) grafts. Installing a dialyzer or a filter into the perfusion circuit to maintain the perfusate condition has some advantages. However, the consequences of purification perfusate during subnormothermic machine perfusion (SNMP) remain unexplained. In this study, the effects of initial purification perfusate with simple method of replacing the first 0.5-L perfusate during SNMP were investigated to consider installation effect of the filter or the dialyzer. Porcine liver grafts, which have 60-min warm ischemia time, were procured to imitate the DCD graft condition. Purified SNMP (PSNMP) results were compared with simple cold storage and conventional SNMP. In PSNMP, initial perfusate of 0.5 L was removed to substitute for purification. After preservation process, the preserved grafts were reperfused with diluted autologous blood for 2 h under normothermic machine perfusion condition to evaluate the liver function using an isolated reperfusion model. The vascular pressures, enzyme release rates and the metabolic indexes during reperfusion were analyzed. The pressures in the hepatic artery after reperfusion 60 min were significantly lower in PSNMP group compared with cold storage (CS) and SNMP groups. In addition, lactate dehydrogenase and alkaline phosphatase were significantly lower after PSNMP than after the CS or SNMP. Also, the metabolic indexes of hyaluronic acid and lactate were significantly decreased by purifying the perfusate in MP preservation than in CS or SNMP. The effectiveness of initial purification perfusate during SNMP was investigated.
Subject(s)
Liver Transplantation/methods , Organ Preservation/methods , Animals , Death, Sudden, Cardiac , Perfusion/methods , Swine , Warm IschemiaABSTRACT
The recent clinical application of perfusion technology for the machine preservation of donation after cardiac death (DCD) grafts has some advantages. Oxygenation has been proposed for the preservation of DCD liver grafts. The aim of this study is to clarify whether the use of HbV-containing preservation solution during the subnormothermic machine perfusion (SNMP) of the liver graft improves the graft function of DCD porcine livers in an ex vivo reperfusion model. Pig livers were excised after 60 minutes of warm ischemic time and were preserved under one of three preservation conditions for 4 hours. The preservation conditions were as follows: 4°C cold storage (CS group; N = 5), Hypothermic machine preservation (HMP) with UW gluconate solution (HMP group; N = 5), SNMP (21°C) with UW gluconate solution (SNMP group; N = 5), SNMP (21°C) with HbVs (Hb; 1.8 mg/dl) perfusate (SNMP+HbV group; N = 5). Autologous blood perfusion was performed for 2 hours in an isolated liver reperfusion model (IRM). The oxygen consumption of the SNMP and SNMP+HbV group was higher than the HMP groups (p < 0.05). During the reperfusion, the AST level in the SNMP+HbV group was lower than that in the CS, HMP and SNMP groups. The changes in pH after reperfusion was significantly lower in SNMP+HbV group than CS and HMP groups. The ultrastructural findings indicated that the mitochondria of the SNMP+HbV group was well maintained in comparison to the CS, HMP and SNMP groups. The SNMP+HbVs preservation solution protected against metabolic acidosis and preserved the liver function after reperfusion injury in the DCD liver.
Subject(s)
Hemoglobins/chemistry , Liver/pathology , Models, Animal , Organ Preservation/methods , Oxygen/chemistry , Adenosine/chemistry , Allopurinol/chemistry , Animals , Aspartate Aminotransferases/metabolism , Female , Glutathione/chemistry , Hemoglobins/metabolism , Hepatic Artery/physiology , Humans , Hydrogen-Ion Concentration , Insulin/chemistry , Lactic Acid/metabolism , Liver/metabolism , Liver Transplantation , Mitochondria/ultrastructure , Organ Preservation/instrumentation , Organ Preservation Solutions/chemistry , Oxygen/metabolism , Oxygen Consumption , Raffinose/chemistry , Swine , TemperatureABSTRACT
BACKGROUND: Bile, which is secreted by the liver, is essential for digesting fat and maintaining homeostasis. Although the rheology of bile is very important to its flow, its extensional viscosity has not been studied. OBJECTIVE: We investigated the stretching of bile and evaluated its extensional viscosity using a filament-breakup device. METHODS: A liquid bridge formed between the endplates of the filament-breakup device. The midpoint diameter of the bridge was recorded by a laser micrometer. The filament self-thinning was captured by a high-resolution, high-speed camera. We used the liquid bridge diameter to evaluate the extensional viscosity of bile. RESULTS: All samples show shear-thinning and viscoelasticity. Bile sediment has much greater viscosity and stretches more than bile solution. Filament thinning takes a long time for bile sediment and a short time for solution. The global function for the mid-filament diameter can be used to predict the evolution of the filament diameter of the liquid bridge and the trend of the extensional viscosity of bile. CONCLUSIONS: The extensional viscosity of bile is much greater than its shear viscosity. Because mucus concentration makes bile sediment more viscous than the solution, bile viscosity can increase sharply, thereby increasing risks of such diseases as gallstones and sludge buildup in the biliary system.
Subject(s)
Bile/chemistry , Humans , Rheology , Shear Strength , Tensile Strength , ViscosityABSTRACT
BACKGROUND Machine perfusion techniques offer a solution to the serious organ shortage. However, to assess the effects of machine perfusion, many detailed studies are required. In this study, an ex vivo reperfusion model using diluted autologous blood was confirmed to evaluate the utility of machine preservation for livers donated after cardiac death (DCD). In particular, beneficial effects of the oxygenated hypothermic machine perfusion (HMP) for DCD porcine livers are evaluated. MATERIAL AND METHODS Porcine livers were procured under warm ischemia time (WIT) of 60 min. The livers were preserved by hypothermic machine perfusion (HMP) or static cold storage (CS) for 4 h. After the preservation, the livers were perfused for 2 h using the ex vivo reperfusion model with diluted blood oxygenated by a membrane oxygenator at 35-38°C. RESULTS At 2 h of ex vivo reperfusion with 60 min of warm ischemic time (WIT), the portal vein pressure for CS was higher than HMP (18.8±15.9 vs. 7.5±3.9 [mmHg] in 60 min). Furthermore, LDH in CS was higher than HMP (528.5±149.8 vs. 194.1±32.2 [IU/L/100 g liver] in 60 min. P<0.05). Lactate after CS (60) was significantly higher than HMP (60) (8.67±0.39 vs. 5.68±0.60 [mmol/L] at 60 min. p<0.01). CONCLUSIONS The ex vivo reperfusion model can be used to evaluate the utility of machine perfusion. Advantages of HMP for DCD livers are evaluated with this model.