Home About us Contact
|
Home About us Contact | ||
|
|
||
Urea Production (urea + production)
Selected AbstractsDevelopment of an Improved Technique for the Perfusion of the Isolated Caudal Lobe of Sheep LiverEXPERIMENTAL PHYSIOLOGY, Issue 5 2000A. M. Ali The study was designed to develop an improved technique for perfusing the isolated caudal lobe of sheep liver. Twenty caudal lobes were perfused for 3-4 h, in a non-recirculating mode, with Krebs-Henseleit bicarbonate buffer. The perfusion system was designed to give a constant flow. The hepatic viability and functional normality of the perfused lobe were assessed by measuring the perfusion flow rate, pH, K+ efflux, O2 uptake, substrate uptake, gluconeogenesis from propionate and amino acids, and ureagenesis from ammonia and amino acids. Liver tissue was sampled for histological examination, as well as for the determination of liver glycogen and wet: dry weight ratio. The perfusion flow rate and pH were both stable throughout the perfusion. The potassium concentration in the effluent perfusate did not increase during the perfusion, suggesting that there was no loss of viability or hypoxia. The perfused lobe extracted more than 50% of the O2 supply. The rate of oxygen consumption was comparable to the rate reported in vivo. The initial glycogen content was reduced by about 40% after 4 h perfusion. The wet: dry weight ratio was 3.6, consistent with the absence of tissue oedema. Urea production was stimulated when NH4Cl (0.3 mM) was added to the medium but there was no significant increase in urea release when alanine (0.15 mM), glutamine (0.2 mM) or lysine (0.2 mM) was added. Urea production, however, increased by about 171% when a physiological mixture of amino acids was added. Propionate (0.5 mM), alanine and glutamine stimulated glucose production but not lysine or the complete amino acid mixture. Glutamine release was lower than that reported in the rat liver. Changing the direction of flow also revealed an apparent difference between livers from sheep and rats in their metabolism of ammonia. The improved technique offers a simple practical and inexpensive approach to many problems in ruminant physiology and nutritional biochemistry. [source] Culture and Characterization of Human Hepatocytes Obtained after Graft Reduction for Liver Transplantation: A Reliable Source of Cells for a Bioartificial LiverARTIFICIAL ORGANS, Issue 7 2004Mariana Barbich Abstract:, This article describes results obtained when human liver cells obtained from reduced grafts are cultured in a chemically defined medium. Remnants of livers after reduction for pediatric transplantation were processed by a multiple cannulation system through the existing vasculature, which allowed the homogeneous perfusion of collagenase. The graft weight ranged between 55 and 1000 g (median value: 145.6 g). The yield ranged between 0.13 × 106 and 38 × 106 cells/g of tissue (median value 14.73 × 106 cells/g), and the viability was 61.17 ± 27.43%. The total number of cells ranged between 57.6 × 106 and 12 150 × 106 cells (median value: 740 × 106 cells). Cells were cultured for 30 days. Albumin synthesis was observed during the first 2 weeks, with a peak value at day 6 (27.85 ± 1.77 µg/mL). Urea production was detected during the first week (peak value at day 6: 17.12 ± 2.11 mg/dL). Light microscopy showed the presence of cells in a monolayer. Biliary pigments were observed at day 20. By immunohistochemistry, positive cells for albumin, for hepatocyte marker, cytokeratin 19, CD 34, CD 68, and for alpha actin for smooth muscle, were observed. Our results showed that hepatocytes obtained from reduced liver grafts are easily cultured and are able to maintain viability and functionality in vitro. This alternative source of human cells maintained under controlled culture conditions may play an important role in the development of a bioartificial liver. [source] NeoHepatocytes From Alcoholics and Controls Express Hepatocyte Markers and Display Reduced Fibrogenic TGF-,/Smad3 Signaling: Advantage for Cell Transplantation?ALCOHOLISM, Issue 4 2010Sabrina Ehnert Background:, Liver transplantation is the only definitive treatment for end stage liver disease. Donor organ scarcity raises a growing interest in new therapeutic options. Recently, we have shown that injection of monocyte-derived NeoHepatocytes can increase survival in rats with extended liver resection. In order to apply this technology in humans with chronic liver diseases in an autologous setting, we generated NeoHepatocytes from patients with alcoholic liver disease and healthy controls and compared those to human hepatocytes. Methods:, We generated NeoHepatocytes from alcoholics with Child A and B cirrhosis and healthy controls. Hepatocytes marker expression and transforming growth factor (TGF)-, signaling was investigated by RT-PCR, Western blot, immunofluorescent staining, and adenoviral reporter assays. Glucose and urea was measured photometrically. Phase I and II enzyme activities were measured using fluorogenic substrates. Neutral lipids were visualized by Oil Red O staining. Results:, There was no significant difference in generation and yield of NeoHepatocytes from alcoholics and controls. Hepatocyte markers, e.g., cytokeratin18 and alcohol dehydrogenase 1, increased significantly throughout differentiation. Glucose and urea production did not differ between alcoholics and controls and was comparable to human hepatocytes. During differentiation, phase I and II enzyme activities increased, however remained significantly lower than in human hepatocytes. Fat accumulation was induced by treatment with insulin, TGF-, and ethanol only in differentiated cells and hepatocytes. TGF-, signaling, via Smad transcription factors, critically required for progression of chronic liver disease, was comparable among the investigated cell types, merely expression of Smad1 and -3 was reduced (,30 and ,60%) in monocytes, programmable cells of monocytic origin, and NeoHepatocytes. Subsequently, expression of TGF-, regulated pro-fibrogenic genes, e.g., connective tissue growth factor and fibronectin was reduced. Conclusions:, Generation of NeoHepatocytes from alcoholics, displaying several features of human hepatocytes, offers new perspectives for cell therapeutic approaches, as cells can be obtained repeatedly in a noninvasive manner. Furthermore, the autologous setting reduces the need for immunosuppressants, which may support recovery of patients which are declined for liver transplantation. [source] Mechanical Dissociation of Swine Liver to Produce Organoid Units for Tissue Engineering and In Vitro Disease ModelingARTIFICIAL ORGANS, Issue 1 2010Katayun Irani Abstract The complex intricate architecture of the liver is crucial to hepatic function. Standard protocols used for enzymatic digestion to isolate hepatocytes destroy tissue structure and result in significant loss of synthetic, metabolic, and detoxification processes. We describe a process using mechanical dissociation to generate hepatic organoids with preserved intrinsic tissue architecture from swine liver. Oxygen-supplemented perfusion culture better preserved organoid viability, morphology, serum protein synthesis, and urea production, compared with standard and oxygen-supplemented static culture. Hepatic organoids offer an alternative source for hepatic assist devices, engineered liver, disease modeling, and xenobiotic testing. [source] Effects of glucose and insulin on HepG2-C3A cell metabolismBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Vidya V. Iyer Abstract HepG2, hepatocellular carcinoma cells, are used in drug toxicity studies and have also been explored for bioartificial livers. For these applications, the cells are under variable levels of nutrients and hormones, the effects of which on metabolism are poorly understood. In this study, HepG2-C3A cells were cultured under varying levels of glucose (high, low, and glucose-free) and insulin (without and with physiological levels of insulin) for 5 days. Cell growth was found to be comparable between high and low glucose media and lowest for glucose-free medium. Several features of central metabolism were affected profoundly by the medium glucose levels. Glucose consumption was greater for low glucose medium compared to high glucose medium, consistent with known glucose feedback regulation mechanisms. Urea productivity was highest in glucose-free medium. Further, it was seen that lactate acted as an alternative carbon source in the absence of glucose, whereas it acted as a sink for the high and low glucose media. Using a metabolic network flexibility analysis (MNFA) framework with stoichiometric and thermodynamic constraints, intracellular fluxes under varying levels of glucose and insulin were evaluated. The analysis indicates that urea production in HepG2-C3A cells arises via the arginase II pathway rather than from ammonia detoxification. Further, involvement of the putrescine metabolism with glutamine metabolism caused higher urea production in glucose-free medium consistent with higher glutamine uptake. MNFA indicated that in high and low glucose media, glycolysis, glutaminolysis, and oxidative phosphorylation were the main sources of energy (NADH, NADPH, and ATP). In the glucose-free medium, due to very low glycolytic flux, higher malate to pyruvate glutaminolytic flux and TCA cycle contributed more significantly to energy metabolism. The presence of insulin lowered glycerol uptake and corresponding fluxes involved in lipid metabolism for all glucose levels but otherwise exerted negligible effect on metabolism. HepG2-C3A cells thus show distinct differences from primary hepatocytes in terms of energy metabolism and urea production. This knowledge can be used to design media supplements and metabolically engineer cells to restore necessary hepatic functions to HepG2-C3A cells for a range of applications. Biotechnol. Bioeng. 2010;107: 347,356. © 2010 Wiley Periodicals, Inc. [source] Application of Multivariate Analysis to Optimize Function of Cultured HepatocytesBIOTECHNOLOGY PROGRESS, Issue 2 2003Christina Chan Understanding the metabolic and regulatory pathways of hepatocytes is important for biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. To characterize intermediary metabolism in the hepatocytes, metabolic flux analysis (MFA) was applied to elucidate the changes in intracellular pathway fluxes of primary rat hepatocytes exposed to human plasma and to provide a comprehensive snapshot of the hepatic metabolic profile. In the current study, the combination of preconditioning and plasma supplementation produced distinct metabolic states. Combining the metabolic flux distribution obtained by MFA with methodologies such as Fisher discriminant analysis (FDA) and partial least squares or projection to latent structures (PLS) provided insights into the underlying structure and causal relationship within the data. With the aid of these analyses, patterns in the cellular response of the hepatocytes that contributed to the separation of the different hepatic states were identified. Of particular interest was the recognition of distal pathways that strongly correlated with a particular hepatic function. The hepatic functions investigated were intracellular triglyceride accumulation and urea production. This study illustrates a framework for optimizing hepatic function and a possibility of identifying potential targets for improving hepatic functions. [source] Culture and in vitro hepatogenic differentiation of placenta-derived stem cells, using placental extract as an alternative to serumCELL PROLIFERATION, Issue 5 2010K. S. Shin Objectives:, Translational research using adult stem cells derived from various tissues has been highlighted in cell-based therapy. However, there are many limitations to using conventional culture systems of adult stem cells for clinically applicability, including limited combinations of cytokines and use of nutrients derived from animals. Here, we have investigated the effects of placental extract (PE) for culture of placenta-derived stem cells (PDSCs) as well as their potential for hepatogenic differentiation. Materials and methods:, Placental extract, extracted using water-soluble methods, was used as a supplement for culture of PDSCs. Cell viability was determined using the MTT assay, and cytokine assay was performed using Luminex assay kit. Gene expression, indocyanine green (ICG) up-take, PAS (Periodic Acid-Schiff) staining and urea production were also analysed. Results:, The placental extract contained several types of cytokine and chemokine essential for maintenance and differentiation of stem cells. Expression of stemness markers in PDSCs cultured with PE is no different from that of PDSCs cultured with foetal bovine serum (FBS). After hepatogenic differentiation, expression patterns for hepatocyte-specific markers in PDSCs cultured with PE were consistent and potential for hepatogenic differentiation of PDSCs cultured with PE was similar to that of PDSCs cultured with FBS, as shown by PAS staining and urea production assays. Conclusions:, Our findings revealed that placental extract could be used as a new component for culture of adult stem cells, as well as for development of human-based medium, in translational research for regenerative medicine. [source] | |||||||||||||