Rate Of Urea Synthesis Research Articles

Boosting the Urea Synthesis Rate on Ni Single-Atom Catalysts: The Impact of Acetonitrile Electrolyte in the Tandem CO2 Reduction/Nucleophilic Addition Reaction

Boosting the Urea Synthesis Rate on Ni Single-Atom Catalysts: The Impact of Acetonitrile Electrolyte in the Tandem CO₂ Reduction/Nucleophilic Addition Reaction

Bimetal selenide NiSe/ZnSe heterostructured nanoparticals decorated porous g-C3N4 nanosheets to boost H2 evolution and urea synthesis

Promoting the separation and transfer of photocarries is very critical in designing an effective photocatalyst. In this study, a novel bimetal selenide ZnxNi1−xSe with NiSe/ZnSe heterostructure prepared by one-step selenization to construct ternary catalytic system NiSe/ZnSe/g-C3N4 (ZnxNi1−xSe/g-C3N4). Consequently, the photocatalytic activity of ZnxNi1−xSe/g-C3N4 is greatly enhanced and higher than that of binary ZnSe/g-C3N4 or NiSe/g-C3N4. The hydrogen production rate of 7 % Zn0.7Ni0.3Se/g-C3N4 reaches 410.15 μmol h−1 g−1, which is 34 times of pure g-C3N4 and 1.5 times of 1 % Pt/g-C3N4. For urea synthesis, 10 % Zn0.7Ni0.3Se/g-C3N4 show the highest urea synthesis rate of 1.12 μmol h−1 g−1, which is about 6 times of pure g-C3N4. The intrinsic mechanism analysis indicates that bimetal selenide NiSe/ZnSe heterojunction plays two roles in ZnxNi1−xSe/g-C3N4 system. Both ZnSe and g-C3N4 forms a type II heterojunction to effectively separate photocarriers. Meanwhile, NiSe provides hydrogen evolution active sites due to its smaller H adsorption free energy, making hydrogen more easily generated and released on it. The synergistic effect of ZnSe/g-C3N4 type II heterojunction and NiSe co-catalyst is more conducive to the separation and transfer of photocarries, thereby resulting in a remarkably enhanced activity. This work provides a unique construction method to design an effective ternary photocatalysts with a double stimulative effect.

Improved Hepatogenic Differentiation of Human Adipose Tissue‐derived MSCs Using a Silicone‐based Cell Culture System

Conventional two‐dimensional as well as established three‐dimensional cell culture systems exhibit a high modulus of elasticity, yet, without representing the stiffness of human organs. Previously, we established a protocol for the culture of primary rat hepatocytes on a silicone of low stiffness, which sustained their physiological functions. In terms of the development of human‐based tests to evaluate the potential risk of novel drugs, the method was adjusted for use of human adipose tissue‐derived mesenchymal stromal cells (hAT‐MSC). Aim of the study was to improve hepatogenic differentiation of hAT‐MSC on fibronectin‐coated 3D silicone scaffolds with low stiffness (E‐modulus approx. 250 kPA) mimicking the stiffness of human liver including surface modifications to increase hydrophilic properties.hAT‐MSCs were cultured on 3D silicone scaffolds. Hepatogenic differentiation was initiated by the 2 step protocol. The capability of the hepatocyte‐like cells to store glycogen, to synthesize urea and albumin as well as the enzyme activities of CYP1A1 and Cyp2B1/2 were analysed and compared to the conventional 2D cell culture and hepatogenic differentiation on polystyrene (E‐modulus 103 MPa).hAT‐MSC attached to the 3D silicone scaffolds and formed cell clusters. 10 days after starting the hepatogenic differentiation until the end of the culture on day 24, the cells on 3D silicone scaffolds displayed more intensive PAS staining for glycogen as compared to the cells on 2D cultures indicating improved glycogen storage. The urea synthesis rate [nmol/10.000 cells/24h] of these cells was significant higher as compared to cells on 2D culture throughout the culture period, though declining with ongoing culture; 10 d: 2D vs 3D 0.05±0.01 vs 1.68±0.05 and 24 d: 0.03±0.007 vs 0.4±0.04, respectively. The albumin synthesis rate [pg/10.000 cells/24h] was significant higher in cells differentiated for 24 d as compared to undifferentiated cells: 48.5±11 vs 7.4±1.9. Enzyme activity of CYP1A1 after 24 d was 3‐fold higher in cells differentiated on 3D silicone scaffolds as compared to cells cultivated on 2D and the enzyme activity of CYP2B1/2 was 12‐fold higher.With these results, medically approved silicone treated with plasma was identified to improve hepatogenic differentiation of hAT‐MSC, likely by providing the appropriate three‐dimensional growth conditions. Thus, the silicone scaffolds represent an important tool for the development of cell cultures featuring organ‐like stiffness and improving physiological functions of hepatogenic differentiated MSC.Support or Funding InformationThe study was supported by the European Regional Development Fund and the Free State of Saxony (SAB) 100282466 to J.H. and P.S.

A revised representation of urea and ammonia nitrogen recycling and use in the Molly cow model

Accurately predicting nitrogen (N) digestion, absorption, and metabolism will allow formulation of diets that more closely match true animal needs from a broad range of feeds, thereby allowing efficiency of N utilization and profit to be maximized. The objectives of this study were to advance representations of N recycling between blood and the gut and urinary N excretion in the Molly cow model. The current work includes enhancements (1) representing ammonia passage to the small intestine; (2) deriving parameters defining urea synthesis and ruminal urea entry rates; (3) adding representations of intestinal urea entry, microbial protein synthesis in the hindgut, and fecal urea-N excretion; and (4) altering existing urinary N excretion equations to scale with body weight and adding purine derivatives as a component of urinary N excretion. After the modifications, prediction errors for ruminal outflows of total N, microbial N, and nonammonia, nonmicrobial N were 29.8, 32.3, and 26.2% of the respective observed mean values. Prediction errors of each were approximately 7 percentage units lower than the corresponding values before model modifications and fitting due primarily to decreased slope bias. The revised model predicted ruminal ammonia and blood urea concentrations with substantially decreased overall error and reductions in slope and mean bias. Prediction errors for gut urea-N entry were decreased from 70.5 to 26.7%, which was also a substantial improvement. Adding purine derivatives to urinary N predictions improved the accuracy of predictions of urinary N output. However, urinary urea-N excretion remains poorly predicted with 69.0% prediction errors, due mostly to overestimated urea-N entry rates. Adding representations of undigested microbial nucleic acids, microbial protein synthesized in the hindgut, and urea-N excretion in feces decreased prediction errors for fecal N excretion from 21.1 to 17.1%. The revised model predicts that urea-N entry into blood accounts for approximately 64% of dietary N intake, of which 64% is recycled to the gut lumen. Between 48 and 67% of the urea recycled to the gut flows into the rumen largely depending on diet, which accounts for 29 to 54% of total ruminal ammonia production, and 65 to 76% of this ammonia-N is captured in microbial protein, which represents 17% of N intake. Based on model simulations, feeding a diet with moderately low crude protein and high rumen-undegradable protein could increase apparent ruminal N efficiency by 20%.

Primary Hepatocytes Cultured on a Fiber-Embedded PDMS Chip to Study Drug Metabolism.

In vitro drug screening using reliable and predictable liver models remains a challenge. The identification of an ideal biological substrate is essential to maintain hepatocyte functions during in vitro culture. Here, we developed a fiber-embedded polydimethylsiloxane (PDMS) chip to culture hepatocytes. Hepatocyte spheroids formed in this device were subjected to different flow rates, of which a flow rate of 50 μL/min provided the optimal microenvironment for spheroid formation, maintained significantly higher rates of albumin and urea synthesis, yielded higher CYP3A1 (cytochrome P450 3A1) and CYP2C11 (cytochrome P450 2C11) enzyme activities for metabolism, and demonstrated higher expression levels of liver-specific genes. In vitro metabolism tests on tolbutamide and testosterone by hepatocytes indicated predicted clearance rates of 1.98 ± 0.43 and 40.80 ± 10.13 mL/min/kg, respectively, which showed a good in vitro–in vivo correspondence. These results indicate that this system provides a strategy for the construction of functional engineered liver tissue that can be used to study drug metabolism.

Testosterone prevents protein loss via the hepatic urea cycle in human.

The urea cycle is a rate-limiting step for amino acid nitrogen elimination. The rate of urea synthesis is a true indicator of whole-body protein catabolism. Testosterone reduces protein and nitrogen loss. The effect of testosterone on hepatic urea synthesis in humans has not been studied. To determine whether testosterone reduces hepatic urea production. An open-label study. Eight hypogonadal men were studied at baseline, and after two weeks of transdermal testosterone replacement (Testogel, 100 mg/day). The rate of hepatic urea synthesis was measured by the urea turnover technique using stable isotope methodology, with 15N2-urea as tracer. Whole-body leucine turnover was measured, from which leucine rate of appearance (LRa), an index of protein breakdown and leucine oxidation (Lox), a measure of irreversible protein loss, were calculated. Testosterone administration significantly reduced the rate of hepatic urea production (from 544.4 ± 71.8 to 431.7 ± 68.3 µmol/min; P < 0.01), which was paralleled by a significant reduction in serum urea concentration. Testosterone treatment significantly reduced net protein loss, as measured by percent Lox/LRa, by 19.3 ± 5.8% (P < 0.05). There was a positive association between Lox and hepatic urea production at baseline (r2 = 0.60, P < 0.05) and after testosterone administration (r2 = 0.59, P < 0.05). Testosterone replacement reduces protein loss and hepatic urea synthesis. We conclude that testosterone regulates whole-body protein metabolism by suppressing the urea cycle.

Bioinspired hollow fiber membrane bioreactor using human liver microtissue spheroids

Event Abstract Back to Event Bioinspired hollow fiber membrane bioreactor using human liver microtissue spheroids Loredana De Bartolo1, Haysam M. Ahmed1, 2, Simona Salerno1, Shervin Khakpour1, 2, Sabrina Morelli1 and Lidietta Giorno1 1 National Research Council of Italy - CNR, Institute on Membrane Technology, Italy 2 University of Calabria, Department of Environmental and Chemical Engineering, Italy Introduction: The development of bioartificial devices that are able to favour the liver reconstruction and to modulate cell behaviour is an important challenge in liver tissue engineering. As strategy we realized liver microtissue spheroids in a crossed hollow fiber membrane bioreactor, which was developed for the in vitro long-term perfusion. The bioreactor consists of two bundles of hollow fiber (HF) polyethersulfone (PES) membranes cross-assembled in alternating manner. One bundle of HF membranes provides cells nutrients and metabolites whereas the other HF bundle removes catabolites from cell compartment mimicking in this way the in vivo arterious and venous blood vessels. The combination of these two fiber set creates three compartments: two intraluminal compartments of PES HF in which the medium flows and one extraluminal compartment represented by extracapillary network formed by the fibers in which spheroids are cultured. Materials and Methods: The bioreactor consists of two bundles of PES HF membranes cross-assembled in alternating manner and potted with polyurethane adhesive within glass housing. The membranes were previously characterised in order to establish the physico-chemical, structural and transport properties[1]. The oxygenated medium enters to the membrane bioreactor with a flow rate Qf of 1.5 mL/min that was set on the basis of average retention time. Its fluid dynamics were characterized in terms of cumulative residence time distribution (RTD). Spheroids of primary human hepatocytes were realized in agarose mold and then cultured in the extralumen compartment of the bioreactor. The oxygen uptake, urea synthesis and albumin production rates as well as biotransformation functions were evaluated with time by using specific assays[2]. Numerical analysis of the mass transfer model for oxygen, urea and albumin concentration was performed using COMSOL Multiphysics. Results and Discussion: The geometry of the bioreactor ensured optimal perfusion conditions, which plaid a crucial role together with the surface and transport properties of the HF membranes in guiding the self-assembling of liver microtissue spheroids. Indeed, adjacent spheroids fused in the extracapillary space forming liver microtissues (Fig. 1). The liver microtissue spheroids expressed liver specific functions in terms of urea synthesis, albumin production and diazepam biotransformation at high levels up to 2 weeks. The liver specific functions of the bioreactor were significantly higher with respect to those of the batch system. Furthermore, the bioreactor provided an adequate oxygenation of the liver microtissue spheroids maintaining oxygen concentration levels well above the in vivo liver periportal zone concentration. Conclusion: The bioreactor thanks to its fluid dynamics and mass transfer properties of the fibers allowed to promote the formation liver microtissue spheroids with high specific functionality.

Hepatic differentiation of rat induced pluripotent stem cells in vitro.

To show the efficient generation of hepatocyte-like cells (HLCs) differentiated from the induced pluripotent stem cells (iPSCs) of rats. Hepatic differentiation was achieved using a three-step protocol with several growth factors. First, rat iPSCs were differentiated into definitive endoderm cells using Activin A and Wnt3a treatment. Then fibroblast growth factor 4 and bone morphogenetic protein 2 were added to the culture medium and used to induce hepatic differentiation. Finally, hepatocyte growth factor, Oncostatin M and dexamethasone were used for hepatic maturation. The liver-related markers and functions of HLCs were assessed at the gene and protein levels. After endodermal induction, the differentiated cells expressed endodermal markers forkhead box protein A2 and SRY-box containing gene 17 at the mRNA and protein levels. After 20 d of culture, the iPSCs were differentiated into HLCs. These differentiated cells expressed hepatic markers including α-fetoprotein, albumin CK8, CK18, CK19, and transcription factor HNF-4α. In addition, the cells expressed functional proteins such as α1-antitrypsin, cytochrome P450 1A2 and CYP 3A4. They acted like healthy hepatic cells, storing glycogen and taking up indocyanine green and low-density lipoproteins. Also, the rates of urea synthesis (20 d 1.202 ± 0.080 mg/dL vs 0 d 0.317 ± 0.021 mg/dL, P < 0.01) and albumin secretion (20 d 1.601 ± 0.102 mg/dL vs 0 d 0.313 ± 0.015 mg/dL, P < 0.01) increased significantly as differentiation progressed. Rat iPSCs can differentiate into HLCs rapidly and efficiently. These differentiated cells may be an attractive resource for treatment of end-stage liver disease.

A glycosaminoglycan based, modular tissue scaffold system for rapid assembly of perfusable, high cell density, engineered tissues.

The limited ability to vascularize and perfuse thick, cell-laden tissue constructs has hindered efforts to engineer complex tissues and organs, including liver, heart and kidney. The emerging field of modular tissue engineering aims to address this limitation by fabricating constructs from the bottom up, with the objective of recreating native tissue architecture and promoting extensive vascularization. In this paper, we report the elements of a simple yet efficient method for fabricating vascularized tissue constructs by fusing biodegradable microcapsules with tunable interior environments. Parenchymal cells of various types, (i.e. trophoblasts, vascular smooth muscle cells, hepatocytes) were suspended in glycosaminoglycan (GAG) solutions (4%/1.5% chondroitin sulfate/carboxymethyl cellulose, or 1.5 wt% hyaluronan) and encapsulated by forming chitosan-GAG polyelectrolyte complex membranes around droplets of the cell suspension. The interior capsule environment could be further tuned by blending collagen with or suspending microcarriers in the GAG solution These capsule modules were seeded externally with vascular endothelial cells (VEC), and subsequently fused into tissue constructs possessing VEC-lined, inter-capsule channels. The microcapsules supported high density growth achieving clinically significant cell densities. Fusion of the endothelialized, capsules generated three dimensional constructs with an embedded network of interconnected channels that enabled long-term perfusion culture of the construct. A prototype, engineered liver tissue, formed by fusion of hepatocyte-containing capsules exhibited urea synthesis rates and albumin synthesis rates comparable to standard collagen sandwich hepatocyte cultures. The capsule based, modular approach described here has the potential to allow rapid assembly of tissue constructs with clinically significant cell densities, uniform cell distribution, and endothelialized, perfusable channels.

Waste Nitrogen Metabolism and Excretion in Zebrafish Embryos: Effects of Light, Ammonia, and Nicotinamide

Bony fish primarily excrete ammonia as adults however the persistence of urea cycle genes may reflect a beneficial role for urea production during embryonic stages in protecting the embryo from toxic effects of ammonia produced from a highly nitrogenous yolk. This study aimed to examine the dynamic scope for changes in rates of urea synthesis and excretion in one such species (zebrafish, Danio rerio) by manipulating the intrinsic developmental rate (by alteration of light:dark cycles), as well as by direct chemical manipulation via ammonia injection (to potentially activate urea production) and nicotinamide exposure (to potentially inhibit urea production). Continuous dark exposure delayed development in embryos as evidenced by delayed appearance of hallmark anatomical features (heartbeat, eye pigmentation, body pigmentation, lateral line, fin buds) at 30 and 48 hr post-fertilization, as well by a lower hatching rate compared to embryos reared in continuous light. Both ammonia and urea excretion were similarly effected and were generally higher in embryos continuously exposed to light. Ammonia injection resulted in significant increases (up to fourfold) of urea N excretion and no changes to ammonia excretion rates along with modest increases in yolk ammonia content during 2-6 hr post-injection. Nicotinamide (an inhibitor of urea synthesis in mammals) reduced the ammonia-induced increase in urea excretion and led to retention of ammonia in the yolk and body of the embryo. Our results indicate that there is a relatively rapid and large scope for increases in urea production/excretion rates in developing embryos. Potential mechanisms for these increases are discussed.

Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing

Primary cultures of human hepatocyte spheroids are a promising in vitro model for long-term studies of hepatic metabolism and cytotoxicity. The lack of robust methodologies to culture cell spheroids, as well as a poor characterization of human hepatocyte spheroid architecture and liver-specific functionality, have hampered a widespread adoption of this three-dimensional culture format. In this work, an automated perfusion bioreactor was used to obtain and maintain human hepatocyte spheroids. These spheroids were cultured for 3-4 weeks in serum-free conditions, sustaining their phase I enzyme expression and permitting repeated induction during long culture times; rate of albumin and urea synthesis, as well as phase I and II drug-metabolizing enzyme gene expression and activity of spheroid hepatocyte cultures, presented reproducible profiles, despite basal interdonor variability (n = 3 donors). Immunofluorescence microscopy of human hepatocyte spheroids after 3-4 weeks of long-term culture confirmed the presence of the liver-specific markers, hepatocyte nuclear factor 4α, albumin, cytokeratin 18, and cytochrome P450 3A. Moreover, immunostaining of the atypical protein kinase C apical marker, as well as the excretion of a fluorescent dye, evidenced that these spheroids spontaneously assemble a functional bile canaliculi network, extending from the surface to the interior of the spheroids, after 3-4 weeks of culture. Perfusion bioreactor cultures of primary human hepatocyte spheroids maintain a liver-specific activity and architecture and are thus suitable for drug testing in a long-term, repeated-dose format.

Methionine and protein metabolism in non-alcoholic steatohepatitis: evidence for lower rate of transmethylation of methionine

Hepatic metabolism of methionine is the source of cysteine, the precursor of glutathione, the major intracellular antioxidant in the body. Methionine also is the immediate precursor of SAM (S-adenosylmethionine) the key methyl donor for phosphatidylcholine synthesis required for the export of VLDL (very-low-density lipoprotein) triacylglycerols (triglycerides) from the liver. We have examined the kinetics of methionine, its transmethylation and trans-sulfuration with estimates of whole body rate of protein turnover and urea synthesis in clinically stable biopsy-confirmed subjects with NASH (non-alcoholic steatohepatitis). Subjects with NASH were more insulin-resistant and had significantly higher plasma concentrations of usCRP (ultrasensitive C-reactive protein), TNFα (tumour necrosis factor α) and other inflammatory cytokines. There was no significant effect of insulin resistance and NASH on whole body rate of protein turnover [phenylalanine Ra (rate of appearance)] and on the rate of urea synthesis. The rates of methylation of homocysteine and transmethylation of methionine were significantly lower in NASH compared with controls. There was no difference in the rate of trans-sulfuration of methionine between the two groups. Enteric mixed nutrient load resulted in a significant increase in all the measured parameters of methionine kinetics. Heterozygosity for MTHFR (5,10-methylene-tetrahydrofolate reductase) (677C→T) did not have an impact on methionine metabolism. We speculate that, as a result of oxidant stress possibly due to high fatty acid oxidation, the activity of methionine adenosyltransferase is attenuated resulting in a lower rate of transmethylation of methionine and of SAM synthesis. These results are the first evidence for perturbed metabolism of methionine in NASH in humans and provide a rationale for the development of targeted intervention strategies.

Microfibrillated Cellulose Sheets Coating Oxygen-Permeable PDMS Membranes Induce Rat Hepatocytes 3D Aggregation into Stably-Attached 3D Hemispheroids

Here we report the use of natural, chemically-unmodified, microfibrillated cellulose (MFC) as a matrix for hepatocyte culture. We developed an original cell-culture design composed of a thin 3D-microstructured fibrous substrate consisting of a MFC sheet coating a highly O2-permeable polydimethylsiloxane (PDMS) membrane. The MFC-coated PDMS membranes were obtained according to a simple process where cellulose fibres were deposited from an aqueous suspension on the PDMS surfaces and the films were dried under mild conditions. To enable oxygen diffusion through the membranes, they were assembled on bottomless frames ('O2+' condition). Rat hepatocytes primary-cultured on such MFC-PDMS membranes quickly organized themselves into large hemispherical 3D aggregates which were tightly anchored to the MFC sheets. In contrast, hepatocytes cultured on smooth PDMS membranes in the O2+ system (O2+, PDMS) organized into unstable 2D monolayers which easily detached from the surfaces. Hepatocyte 3D cultures obtained on MFC-PDMS membranes exhibited higher liver-specific functions over a 2-week culture period, as assessed by both the higher albumin secretion and urea synthesis rate. The MFC-PDMS membranes appear suitable for obtaining stably-attached and functional hepatocyte 3D cultures and appear interesting for drug/chemical screenings in a microplate format, but also for microfluidic applications.

In vitro hepatic differentiation of umbilical cord-derived mesenchymal stem cell

Recently hepatic differentiation of mesenchymal stem cells (MSCs) from bone marrow (BM), adipose tissue (AT), umbilical cord blood (UCB), and umbilical cord (UC) has been reported. In this study, the four-step sequential exposure with oncostatin M (OSM) plus trichostatin A (TSA) or OSM plus dimethyl sulfoxide (DMSO) at the final step induced hepatic differentiation of UC-MSCs. As a result, the morphology and protein expression were sequentially changed in the step-dependent manner. And the urea synthesis rates of (OSM plus TSA)- and (OSM plus DMSO)-treated cells on day 21 reached to 23 ± 0.4 and 20 ± 0.5 μg/10 6 cells/day, respectively. The ammonia concentrations 24 h after culture with 1 mM NH 4Cl-supplemented medium dropped to 0.41 ± 0.08 mM (OSM plus TSA) and 0.57 ± 0.05 mM (OSM plus DMSO). Also the ethoxyresorufin O-deethylase (EROD) activities were 3.4-fold (OSM plus TSA) and 2.2-fold (OSM plus DMSO) higher than non-induced controls on day 21. It is thought that TSA and DMSO cause the expression of key genes through epigenetic change. Although sequential exposure with TSA or DMSO induced some liver-specific functions to some extent, the degree of activities are yet lower than those of mature hepatocytes. So it will be necessary to optimize the concentration and exposure time for achieving comparable activities to normal hepatocytes in the future.

Cryopreservation of primary porcine liver cells in an organotypical sandwich model in a clinically relevant flat membrane bioreactor

To overcome the logistical difficulties of continuously supplying freshly-isolated, primary porcine liver cells to bioartificial liver support bioreactors, we developed a cryopreservation method using an organotypical sandwich model in a flat membrane bioreactor (FMB). We measured albumin secretion rate, urea synthesis rate and 7-ethoxy coumarin (ECOD) in long-term cultures of cryopreserved cells (up to 14 days). The albumin secretion rate was 62% that of non-cryopreserved cells at days 11 and 14. The ECOD activity was 54% that of fresh, control cells initially and increased up to 79% by the 14th day. The urea synthesis rate was stable at 60% that of the control. This study showed that cryopreserved cells can recover liver-specific functions. This result has the potential to dramatically expand the clinical application of bioartificial liver supports.

Spontaneous formation of stably-attached and 3D-organized hepatocyte aggregates on oxygen-permeable polydimethylsiloxane membranes having 3D microstructures

In order to enhance the viability and the differentiated functions of primary hepatocytes in cultures, it appears important to have them organized within a three-dimensional (3D) structure which promotes extensive cell-cell contacts, but also to be adequately supplied with oxygen. Here, we report a simple methodology satisfying these two fundamental but sometimes conflicting issues: primary rat hepatocytes were cultured on polydimethylsiloxane (PDMS) membranes with 3D-pillared microstructures with various dimensions, so that the cells could organize themselves around the pillars into various kinds of 3D multicellular aggregates, while being continuously supplied with oxygen by diffusion through the PDMS membrane. As expected, under such conditions, hepatocyte cultures exhibited higher albumin secretion and urea synthesis rates. It appeared then that as the spacing decreased between the pillars, the cells were more stably organized into smaller spherical aggregates and displayed the highest albumin secretion rates. Such a simple design is likely to be included in a new drug/chemical screening system in a practical microplate format, but also appears applicable to microfluidic devices.

Human hepatocyte functions in a crossed hollow fiber membrane bioreactor

An important challenge in liver tissue engineering is the development of bioartificial systems that are able to favour the liver reconstruction and to modulate liver cell behaviour. A crossed hollow fiber membrane bioreactor was developed to support the long-term maintenance and differentiation of human hepatocytes. The bioreactor consists of two types of hollow fiber (HF) membranes with different molecular weight cut-off (MWCO) and physico-chemical properties cross-assembled in alternating manner: modified polyetheretherketone (PEEK-WC) and polyethersulfone (PES), used for the medium inflow and outflow, respectively. The combination of these two fiber set produces an extracapillary network for the adhesion of cells and a high mass exchange through the cross-flow of culture medium. The transport of liver specific products such as albumin and urea together with the transport of drug such as diazepam was modelled and compared with the experimental metabolic data. The theoretical metabolite concentration differed 7.5% for albumin and 5% for urea with respect to experimental data. The optimised perfusion conditions of the bioreactor allowed the maintenance of liver functions in terms of urea synthesis, albumin secretion and diazepam biotransformation up to 18 days of culture. In particular the good performance of the bioreactor was confirmed by the high rate of urea synthesis (28.7 μg/h 10 6 cells) and diazepam biotransformation. In the bioreactor human hepatocytes expressed at high levels the individual cytochrome P450 isoenzymes involved in the diazepam metabolism. The results demonstrated that crossed HF membrane bioreactor is able to support the maintenance of primary human hepatocytes preserving their liver specific functions for all investigated period. This device may be a potential tool in the liver tissue engineering for drug metabolism/toxicity testing and study of disease pathogenesis alternatively to animal experimentation.

Preparation and HL-7702 cell functionality of titania/chitosan composite scaffolds

Titania/chitosan composite scaffolds were prepared through a freeze-drying technique. The composite scaffolds were highly porous with the average pore size of 120-300 microm, and the titania (TiO(2)) powders were uniformly dispersed on the surface of the pore walls. The compressive strength of the composite scaffolds was significantly improved compared to that of pure chitosan scaffolds. Composite scaffold with 0.3 of TiO(2)/chitosan weight ratio showed the maximum compressive strength of 159.7 +/- 21 kPa. Hepatic immortal cell line HL-7702 was used as seeding cells on the scaffolds, and after different culture periods, cell attachment and function was analyzed. HL-7702 cells attached on the pore walls of the scaffolds with the spheroid shape after 1 day of culture, but more cell aggregations formed within the TiO(2)/chitosan composite scaffolds as compared to pure chitosan scaffolds. Liver-specific functions, albumin secretion and urea synthesis were detected using a spectrometric method. The results showed that albumin secretion and urea synthesis rate of HL-7702 cells slightly decreased with the culture time, and there was no significant difference between composite scaffolds and pure chitosan scaffolds. In conclusion, the TiO(2)/chitosan composite scaffolds possessed an improved mechanical strength compared to pure chitosan scaffolds and supported the attachment and functional expression of hepatocyte, implying their potential application in liver tissue engineering.

Effects of hypoxia on the energy status and nitrogen metabolism of African lungfish during aestivation in a mucus cocoon

We examined the energy status, nitrogen metabolism and hepatic glutamate dehydrogenase activity in the African lungfish Protopterus annectens during aestivation in normoxia (air) or hypoxia (2% O(2) in N(2)), with tissues sampled on day 3 (aerial exposure with preparation for aestivation), day 6 (entering into aestivation) or day 12 (undergoing aestivation). There was no accumulation of ammonia in tissues of fish exposed to normoxia or hypoxia throughout the 12-day period. Ammonia toxicity was avoided by increased urea synthesis and/or decreased endogenous N production (as ammonia), but the dependency on these two mechanisms differed between the normoxic and the hypoxic fish. The rate of urea synthesis increased 2.4-fold, with only a 12% decrease in the rate of N production in the normoxic fish. By contrast, the rate of N production in the hypoxic fish decreased by 58%, with no increase in the rate of urea synthesis. Using in vivo (31)P NMR spectroscopy, it was demonstrated that hypoxia led to significantly lower ATP concentration on day 12 and significantly lower creatine phosphate concentration on days 1, 6, 9 and 12 in the anterior region of the fish as compared with normoxia. Additionally, the hypoxic fish had lower creatine phosphate concentration in the middle region than the normoxic fish on day 9. Hence, lowering the dependency on increased urea synthesis to detoxify ammonia, which is energy intensive by reducing N production, would conserve cellular energy during aestivation in hypoxia. Indeed, there were significant increases in glutamate concentrations in tissues of fish aestivating in hypoxia, which indicates decreases in its degradation and/or transamination. Furthermore, there were significant increases in the hepatic glutamate dehydrogenase (GDH) amination activity, the amination/deamination ratio and the dependency of the amination activity on ADP activation in fish on days 6 and 12 in hypoxia, but similar changes occurred only in the normoxic fish on day 12. Therefore, our results indicate for the first time that P. annectens exhibited different adaptive responses during aestivation in normoxia and in hypoxia. They also indicate that reduction in nitrogen metabolism, and probably metabolic rate, did not occur simply in association with aestivation (in normoxia) but responded more effectively to a combined effect of aestivation and hypoxia.

Increased urea synthesis and/or suppressed ammonia production in the African lungfish, Protopterus annectens, during aestivation in air or mud

The objective of this study was to elucidate how the African lungfish, Protopterus annectens, ameliorated ammonia toxicity during 12 or 46 days of aestivation in air or in mud. Twelve days of aestivation in air led to significant increases in contents of urea, but not ammonia, in tissues of P. annectens. The estimated rate of urea synthesis increased 2.7-fold despite the lack of changes in the activities of hepatic ornithine-urea cycle enzymes, but there was only a minor change in the estimated rate of ammonia production. After 46 days of aestivation in air, the ammonia content in the liver decreased significantly and contents of urea in all tissues studied increased significantly, indicating that the fish shifted to a combination of increased urea synthesis (1.4-fold of the day 0 value) and decreased ammonia production (56% of the day 0 value) to defend against ammonia toxicity. By contrast, 12 days of aestivation in mud produced only minor increases in tissue urea contents, with ammonia contents remained unchanged. This was apparently achieved through decreases in urea synthesis and ammonia production (40 and 15%, respectively, of the corresponding day 0 value). Surprisingly, 46 days of aestivation in mud resulted in no changes in tissue urea contents, indicating that profound suppressions of urea synthesis and ammonia production (2.6 and 1.2%, respectively, of the corresponding day 0 value) had occurred. This is the first report on such a phenomenon, and the reduction in ammonia production was so profound that it could be the greatest reduction known among animals. Since fish aestivated in mud had relatively low blood pO(2) and muscle ATP content, they could have been exposed to hypoxia, which induced reductions in metabolic rate and ammonia production. Consequently, fish aestivating in mud had a lower dependency on increased urea synthesis to detoxify ammonia, which is energy intensive, than fish aestivating in air.