Perfused Liver Tissue Research Articles

Monensin induces apoptosis in the liver tissue and primary hepatocytes of chicks

In this study, monensin (MON) was investigated for its apoptotic and genotoxic effects on chick liver tissue and cytotoxic and apoptotic effects on chick primary hepatocytes isolated from perfused liver tissue. Western blotting and real-time PCR (qPCR) were used to determine the apoptotic effect on primary hepatocytes and liver tissue, and the comet assay was used to determine the genotoxic effect on liver tissue. MON decreased cell viability in primary hepatocytes with increasing concentrations. When administered at concentrations of 1 and 10 μM, MON increased the levels of apoptotic p53, caspase-3, and caspase-9, and downregulated the expression of the antiapoptotic survivin, Bcl-xl, and Bcl-2 genes (p < 0.05). Furthermore, 100 and 125 ppm of MON increased caspase-3, caspase-9, and p53 protein levels, and downregulated Bcl-2 expression (p < 0.05). Bcl-xl gene expression was downregulated only in the group that received 125 ppm of MON (p < 0.05). Changes observed in the expression of the survivin gene were insignificant in both groups (p > 0.05). Moreover, 100 and 125 ppm of MON caused comet formation, which is a marker of genotoxicity, in the liver (p < 0.05). Our results indicate that MON induced apoptosis in both primary hepatocytes and liver tissue, and also caused genotoxicity in the liver tissue.

Viability Assessment in Liver Transplantation-What Is the Impact of Dynamic Organ Preservation?

Based on the continuous increase of donor risk, with a majority of organs classified as marginal, quality assessment and prediction of liver function is of utmost importance. This is also caused by the notoriously lack of effective replacement of a failing liver by a device or intensive care treatment. While various parameters of liver function and injury are well-known from clinical practice, the majority of specific tests require prolonged diagnostic time and are more difficult to assess ex situ. In addition, viability assessment of procured organs needs time, because the development of the full picture of cellular injury and the initiation of repair processes depends on metabolic active tissue and reoxygenation with full blood over several hours or days. Measuring injury during cold storage preservation is therefore unlikely to predict the viability after transplantation. In contrast, dynamic organ preservation strategies offer a great opportunity to assess organs before implantation through analysis of recirculating perfusates, bile and perfused liver tissue. Accordingly, several parameters targeting hepatocyte or cholangiocyte function or metabolism have been recently suggested as potential viability tests before organ transplantation. We summarize here a current status of respective machine perfusion tests, and report their clinical relevance.

Developing a partially perfused liver model using caprine liver explant conditioned to the chicken extra-embryonic perfusion system

The Liver is constantly subjected to mechanical, chemical and pathological insults throughout life, as a result of which there is a common occurrence of various liver diseases. Due to the complex nature of liver architecture, it is not possible to mimic the in-vivo conditions beyond a certain limit. Hence, the development of in-vitro and ex-vivo models to study various liver diseases has gained more importance over the last few decades. The present study aims to develop a semi-perfused liver explant model to give an extended lifetime for studying liver pathology and treatment options. Caprine liver tissue explants were sliced, weighed (25 mg) and placed on the area vasculosa of fertilized chicken eggs. Unfertilized eggs were used as controls. After varying time intervals of incubation on area vasculosa of fertilized chicken eggs, the liver slices were subjected to cell viability assay, lactate dehydrogenase assay and Serum glutamic pyruvic transaminase assays. The results indicate that the viability and functional properties of such semi-perfused liver tissue explants holds good up to 6 h. Finally, the liver tissue explants were pre-treated with FBS, with and without anti-fibrotic drugs, and placed on chick embryo area vasculosa up to 6 h. The anti-fibrotic drug-treated semi perfused liver tissue explant showed a decrease in collagen formation as confirmed by histology and western blot. We deem that the use of extra-embryonic vasculature of chicken bed for extending the life of tissue explants will serve as a cost-effective alternative to animal models to understand disease mechanisms under drug treatments.

Equine hepatocytes: isolation, cryopreservation, and applications to invitro drug metabolism studies.

Despite reports of the successful isolation of primary equine hepatocytes, there are no published data regarding the successful cryopreservation of these isolated cells. In this study, a detailed description of the procedures for isolation, cryopreservation, and recovery of equine hepatocytes are presented. Furthermore, the intrinsic clearance (Clint) and production of metabolites for three drugs were compared between freshly isolated and recovered cryopreserved hepatocytes. Primary equine hepatocytes were isolated using a two‐step collagenase perfusion method, with an average cell yield of 2.47 ± 2.62 × 106 cells/g of perfused liver tissue and viability of 84.1 ± 2.62%. These cells were cryopreserved with William's medium E containing 10% fetal bovine serum with 10% DMSO. The viability of recovered cells, after a 30% Percoll gradient, was 77 ± 11% and estimated recovery rate was approximately 27%. These purified cells were used to determine the in vitro Clint of three drugs used in equine medicine; omeprazole, flunixin, and phenylbutazone, via the substrate depletion method. Cryopreserved suspensions gave a comparable estimation of Clint compared to fresh cells for these three drugs as well as producing the same metabolites. This work paves the way for establishing a bank of cryopreserved equine hepatocytes that can be used for estimating pharmacokinetic parameters such as the hepatic metabolic in vivo clearance of a drug as well as producing horse‐specific drug metabolites.

The impact of blood on liver metabolite profiling – a combined metabolomic and proteomic approach

Metabolomics has entered the well-established omic sciences as it is an indispensable information resource to achieve a global picture of biological systems. The aim of the present study was to estimate the influence of blood removal from mice liver as part of sample preparation for metabolomic and proteomic studies. For this purpose, perfused mice liver tissue (i.e. with blood removed) and unperfused mice liver tissue (i.e. containing blood) were compared by two-dimensional gas chromatography time of flight mass spectrometry (GC × GC-TOFMS) for the metabolomic part, and by liquid chromatography tandem mass spectrometry (LC-MS/MS) for the proteomic part. Our data showed significant differences between the unperfused and perfused liver tissue samples. Furthermore, we also observed an overlap of blood and tissue metabolite profiles in our data, suggesting that the perfusion of liver tissue prior to analysis is beneficial for an accurate metabolic profile of this organ.

Scale-dependent mechanical properties of native and decellularized liver tissue

Decellularization, a technique used in liver regenerative medicine, is the removal of all the cellular components from a tissue or organ, leaving behind an intact structure of extracellular matrix. The biomechanical properties of this novel scaffold material are currently unknown and are important due to the mechanosensitivity of liver cells. Characterizing this material is important for bioengineering liver tissue from this decellularized scaffold as well as creating new 3-dimensional mimetic structures of liver extracellular matrix. This study set out to characterize the biomechanical properties of perfused liver tissue in its native and decellularized states on both a macro- and nano-scale. Poroviscoelastic finite element models were then used to extract the fluid and solid mechanical properties from the experimental data. Tissue-level spherical indentation-relaxation tests were performed on 5 native livers and 8 decellularized livers at two indentation rates and at multiple perfusion rates. Cellular-level spherical nanoindentation was performed on 2 native livers and 1 decellularized liver. Tissue-level results found native liver tissue to possess a long-term Young's modulus of 10.5 kPa and decellularized tissue a modulus of 1.18 kPa. Cellular-level testing found native tissue to have a long-term Young's modulus of 4.40 kPa and decellularized tissue to have a modulus of 0.91 kPa. These results are important for regenerative medicine and tissue engineering where cellular response is dependent on the mechanical properties of the engineered scaffold.

Structural characterization of blood group A glycolipids in blood group A1 liver tissue in situ perfused with O blood: the dominating presence of type 1 core chain A antigens

Biochemical studies of organ blood group antigen expression show a mixed pattern originating from both the organ tissue and remaining blood cells trapped in the organ despite in vitro perfusion of the vascular tree. The blood group A glycolipid expression was studied in a unique case in which a human liver had been in situ perfused by recipient blood. A blood group O recipient was re-transplanted with an ABO incompatible A1Le (a - b +) liver. Because of discrepancy in size, liver segments II and III were removed 2 h after re-vascularization. Thereafter, the removed A1 liver segment was physiologically in situ perfused with O blood, eliminating a major part of the donor blood cells/plasma. Total neutral glycolipids were isolated from the liver tissue and separated by high-performance liquid chromatography. Purified glycolipid fractions were stained with anti-A monoclonal antibodies (mAbs) and structurally characterized by mass spectrometry and proton nuclear magnetic resonance (NMR) spectroscopy. Two blood group A reactive glycolipid compounds were isolated. One component had a thin-layer chromatography (TLC) mobility as a six-sugar glycolipid and reacted with mAbs specific for A type 1 mono-fucosyl structures. The second glycolipid fraction migrated as seven-sugar components and reacted with mAbs specific for type 1 difucosyl (ALeb) as well as Leb determinants. Mass spectrometry of the six-sugar component showed a structure similar to a blood group A hexaglycosylceramide with one fucose. Mass spectrometry and proton NMR spectroscopy of the seven-sugar fraction revealed a mixture of blood group Leb hexa- and ALeb hepta-glycosylceramides, respectively. All fractions were non-reactive with antibodies specific for A antigens based on types 3 and 4 core chain structures. In addition, TLC immunostaining of glycolipids isolated from blood group A livers, harvested for organ transplantation but discarded for various reasons, revealed trace amounts of several A glycolipids with a complex pattern. The in situ perfused liver tissue contains blood group A glycolipids based exclusively on type 1 core chains. The secretor gene (Se) codes for a fucosyltransferase acting on all core chain precursors while the H-gene fucosyltransferase only utilizes the type 2 chain precursor. Whether this explains that only A type 1 chain compounds were found has to be established.

Preparation of equine isolated hepatocytes

In this study a detailed description of the equine hepatocyte isolation procedure is presented. Livers were obtained from horses slaughtered at the local slaughterhouse. For blood removal and liver preservation the following steps are suggested: perfusion with the oxygenated HBSS (0–2 °C, with continuous flow of 500–800 ml/min for 3–6 min), protection from ischemia injury by flushing with ice-cold University of Wisconsin Solution (UW, flow rate of 500–800 ml/min), and finally immersion of the liver lobe in UW solution (2 °C) during its transport to the laboratory. For equine isolated hepatocyte preparation a “three-step” perfusion procedure was elaborated: rewarming, chelating and collagenase perfusion. We found optimal cell yield and viability under the following conditions: rewarming with UW (38 °C) for 8–14 min, chelating with calcium free Hanks’ Balanced Salt Solution (HBSS, 38 °C) supplemented with 1 m m ethylene glycol-bis[β-aminoethyl esther]-N,N,N′N′-tetracetic acid at the flow rate of 450 ml/min for 6 min and enzymatic digestion with HBSS supplemented with 0.1% collagenase at 38 °C and 450 ml/min flow rate for 8–27 min. These conditions consistently generated cell harvests of 21×10 6±4.86 cells/g of perfused liver tissue with viability of 82.7%±10.2.

Isolated rat liver perfusion studies with cyclic heptapeptide toxins of Microcystis and Oscillatoria (freshwater cyanobacteria)

Isolated perfused rat livers were used to study the dose-dependent effects of three cyclic heptapetide toxins isolated from Norwegian freshwater bloom samples containing Microcystis aeruginosa, Oscillatoria agardhii var. and Oscillatoria agardhii var. isothrix. The high pressure liquid chromatography (HPLC) purified toxins had an i.p. ld 50 in the rat and mouse of approximately 50, 500 and 1000 μg/kg, respectively. Hepatic insult of the toxins at concentrations of 0.5 – 4.0 times the rat i.p. lethal dose were assessed by monitoring bile flow, accumulation of total protein in the perfusate, release of intracellular enzymes and histopathologic examination of perfused liver tissue. One hundred micrograms of Microcystis toxin produced cessation of bile flow during a 1 hr perfusion period, while the two Oscillatoria toxins required 1000 and 2000 μg of toxin, consistent with their lower ld 50 values. Hepatic cell membranes remained intact during the perfusion since release of enzymes and proteins into the perfusate was similar for toxin treated and control livers, and histopathologic examination of Trypan Blue infused livers revealed exclusion of the dye from the intracellular compartment of the parenchyma. Histopathologic findings for all three toxins showed hepatocellular disassociation that increased with toxin concentration. At the ultrastructural level, all three toxins caused dose-dependent vesiculation of rough endoplasmic reticulum, formation of concentric whorls composed of rough-ER, mitochondrial swelling, large cytoplasmic vacuoles and altered bile canaliculi. These changes were similar to those found for previous in vivo studies using Microcystis cyclic heptapeptides from Scotland and Australia. The Oscillatoria toxins required five to ten times more toxin to produce similar effects as the Microcystis toxin. At the higher concentrations, the Oscillatoria toxins also caused a proliferation of smooth-ER. The isolated perfused rat liver was found to be a good model for studying the hepatocellular effects of different cyclic peptide toxins from cyanobacteria.

The influence of the position of the oxygen dissociation curve on oxygen-dependent functions of the isolated perfused rat liver. II. Studies at different levels of hypoxia induced by decrease of blood flow rate.

The influence of the 2,3-diphosphoglycerate (2,3-DPG) induced displacement of the oxygen dissociation curve (O.D.C.) on the isolated perfused rat liver was studied at different levels of stagnant hypoxia (hypoxia induced by decrease of blood flow rate at constant PO2 and at constant haematocrit). Rat livers were perfused with a medium containing either fresh or 2,3-DPG-depleted erythrocytes (2,3-DPG content: 4.3 +/- 0.4 and 0.6 +/- 0.4 mmol/l erythrocytes, respectively). The difference in oxygen affinity of the red cells did not affect the vascular resistance of the perfused liver tissue. The decrease in oxygen supply brought about by a decrease in blood flow rate resulted in a decrease of bile flow rate and of oxygen consumption. The higher 2,3-DPG content of the fresh erythrocytes was reflected in a higher bile flow rate at all blood flow levels, in a higher oxygen consumption at lower blood flow levels, and in a higher venous PO2 at the higher blood flow levels. Venous PO2, lactate/pyruvate (L/P) ratio and beta-hydroxybutyrate/acetoacetate (betaOH/Acac) ratio were relatively insensitive to stagnant hypoxia and to a difference in the 2,3-DPG content of the erythrocytes. The ATP content of the liver tissue was decreased at the lower blood flow levels. However, the ATP content of the livers perfused with fresh erythrocytes did not differ from that of the livers perfused with 2,3-DPG-depleted erythrocytes.

The redox state of NAD +-NADH systems in rat liver during ketosis, and the so-called “triosephosphate block”

In livers of normal fed rats, 12-h and 96-h starved rats, fat-fed rats, and alloxandiabetic ketotic rats, the concentrations of lactate, pyruvate, ATP, ADP, NAD +, fructose 1,6-diphosphate, triosephosphate, glycogen, and total nitrogen, were determined under conditions in vivo by the freezing-stop technique. Lactate and pyruvate concentrations were also determined in livers from rats which received intraportal infusions of sodium caproate. The liver concentrations of β-hydroxybutyrate, acetoacetate, malate and oxaloacetate were estimated in livers from the 12-h starved and the fat-fed groups. Studies in vitro were carried out with isolated perfused livers of normal and severely diabetic rats for the determination of lactate, pyruvate and total ketone-body concentrations in the medium, and the concentrations of lactate, pyruvate, ATP and ADP in the perfused liver tissue. The perfusion experiments were carried out with or without intraportal infusions of sodium caproate. The results reveal no correlation between the extent of fatty acid oxidation as shown by ketone-body formation and the redox state of the three NAD +-NADH systems in equilibrium with the substrate pairs: lactate-pyruvate, malate-oxaloacetate and β-hydroxybutyrate-acetoacetate. Thus, the increased reduction of the cytoplasmic NAD +-NADH system in livers of severely diabetic rats is not the consequence of enhanced fatty acid oxidation during diabetes. Sodium caproate leads to a significant increase in the uptake of lactate and pyruvate by isolated, perfused livers. While the ATP-ADP ratio was lower in diabetic livers, the ATP concentration showed no decrease. The total concentration of ATP and ADP, as well as the concentration of NAD +, was significantly higher in diabetic livers. The stationary concentrations of fructose 1,6-diphosphate and triosephosphate showed no increase in livers of fat-fed, starved or diabetic rats. No evidence for the existence of a block in glycolysis at the triosephosphate dehydrogenase (EC 1.2.1.12) step could be found, under these conditions.