Dissertations / Theses on the topic 'In Vitro Liver Models'
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Vu, Lucas Trung. "Proteomic Analysis of Three Dimensional Organotypic Liver Models." Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/77033.
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In vitro liver models that closely mimic the in vivo microenvironment are central for understanding hepatic functions and intercellular communication processes. Bottom-up shotgun proteomic analysis of the hepatic cells can lend insight into such processes. This technique employs liquid chromatography-tandem mass spectrometry (LC-MS/MS) for relative quantification of protein abundances by measuring intensities of their corresponding peptides. Organotypic 3D liver models have been developed in our laboratory that consist of hepatocytes and liver sinusoidal endothelial cells (LSECs) separated by a polyelectrolyte multilayer (PEM), which serves as a mimic for the Space of Disse. Each component within these models is easily separable allowing for systematic evaluation of the cells and PEMs. In this study, proteomes of hepatocytes from PEM containing models, cultured with and without LSECs, were compared to those from monolayers. Changes in core metabolism were evaluated among all culture conditions. Overall, all cultures were ketogenic and performed gluconeogenesis. The presence of the PEM led to increases in proteins associated with mitochondrial-based β-oxidation and peroxisomal proteins. The PEMs also limited production of structural proteins, which are linked to dedifferentiation of hepatocytes, suggesting that cell-ECM interactions are essential for maintenance of their liver-like state. The presence of LSECs increased levels of carboxylesterases and other phase I and phase II detoxification enzymes suggesting that intercellular signaling mediates enzyme abundance. Taken together, these results suggest that the cell-cell (from the LSECs) and cell-ECM (from the PEMs) interactions exert different, yet crucial effects, and both are required for the preservation of metabolic liver functions and differentiated phenotypes. Changes in the PEMs as a result of cell culture were also evaluated but exhibited minimal differences at this time point. Proteomes of LSECs monolayers were also characterized. Enzymes related to the metabolism of amino acids, lipids, oxidative phosphorylation and phase I and phase II detoxification processes were all identified in LSECs monolayers highlighting their role in these processes. Characterization of 3DHL LSECs was not possible due to ion suppression resulting from the presence of excess contaminant proteins. Nonetheless, this study provides a foundation in which LSECs from 3D liver models can be compared against in future studies.Ph. D.
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Granitzny, Anne [Verfasser]. "In vitro/ex vivo liver models for the prediction of idiosyncratic drug-induced liver injury / Anne Granitzny." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2017. http://d-nb.info/1150192496/34.
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Tirnitz-Parker, Janina Elke Eleonore. "Primary culture and immortal cell lines as in vitro models to evaluate the role of TWEAK signalling in hepatic oval cells /." Connect to this title, 2007. http://theses.library.uwa.edu.au/adt-WU2008.0039.
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Heslop, James. "Investigating novel methods of enhancing in vitro models of drug induced liver injury." Thesis, University of Liverpool, 2015. http://livrepository.liverpool.ac.uk/2053229/.
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Drug induced liver injury (DILI) is a major cause of patient morbidity and mortality inferring considerable burdens onto healthcare and pharmaceutical sectors. As a consequence, substantial resources are directed towards triaging potentially dangerous new compounds at all stages of drug development. However, despite these efforts, hepatotoxic compounds remain the greatest cause of post-marketing drug withdrawal. One of the major factors preventing efficacious screening of new compounds is the lack of a truly representative in vitro model of hepatotoxicity. This thesis describes our efforts to utilise innovative and emerging techniques to further understand and develop in vitro models of hepatotoxicity. One such technique is the generation of hepatocyte-like cells from induced pluripotent stem cells (iPSCs). iPSC-derived hepatocyte-like cells offer a reproducible, physiologically-relevant, genotypically normal and population-representative model of hepatotoxicity; however, current differentiation protocols are not capable of producing hepatocyte-like cells beyond a relatively immature phenotype, limiting their use for toxicological studies. As part of the cellular reprogramming process the epigenome of the somatic cell undergoes dramatic changes; however, the studies have shown that this ‘resetting’ of the epigenome to a pluripotent state is an imperfect process, resulting in an altered differentiation propensity skewed towards the lineage of origin. We evaluated if using human hepatocytes as the starting cell type and utilising the inherent ‘epigenetic memory’ associated with iPSCs could enhance the maturity of hepatocyte-like cells. Despite a trend towards improvement in phenotype, no significant differences were found between isogenic hepatocyte-derived and fibroblast-derived iPSCs. The further development of hepatocyte-like cells is limited by the inability of current culture systems to adequately support the hepatic phenotype. Once placed into culture, primary human hepatocytes, the gold standard model of hepatotoxicity, quickly lose the metabolic qualities required for modelling drug induced liver injury. Thus, without a culture system which supports the hepatic phenotype, the differentiation of hepatocyte-like cells will remain sub-optimal. Using iTRAQ proteomics we attempted to identify the driving factors responsible for the process of hepatocyte dedifferentiation. Our results identified numerous novel factors, including HSF2, SMARCB1, ZEB1 and FOXO1 which may drive the selective loss of metabolic phenotype. The proteomic assessment of hepatocyte dedifferentiation also highlighted the loss of Nrf2-related proteins during culture. Further investigation of Nrf2 in hepatocytes revealed a potentially negative relationship between Nrf2 induction and the key metabolic enzyme, CYP3A4. Furthermore, Nrf2 gene and protein expression was shown to increase during hepatocyte-like cell differentiation. Taken together, these results suggest that Nrf2 may negatively regulate the hepatic phenotype, potentially preventing the establishment of a mature phenotype during hepatocyte-like cell differentiation. A mechanistic evaluation of Nrf2 during differentiation and dedifferentiation is therefore required to gain a fuller insight into the role it plays in the maintenance and acquisition of the hepatic phenotype. In summary, this thesis presents our contribution to the further understanding, development and enhancement of in vitro hepatotoxicity models, using innovative techniques to assess the impact of epigenetic memory on HLC differentiation, identifying novel influencing factors driving the loss of phenotype in hepatocyte culture systems and evaluating the influence of Nrf2 on the hepatic phenotype during differentiation and dedifferentiation.
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Chang, Robert Chao Sun Wei. "Biofabrication of three-dimensional liver cell-embedded tissue constructs for in vitro drug metabolism models /." Philadelphia, Pa. : Drexel University, 2009. http://hdl.handle.net/1860/3069.
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Regan, Sophie Louise. "In Vitro and In Vivo models for the investigation of drug bioactivation and drug-induced liver injury." Thesis, University of Liverpool, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510967.
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Brabyn, Caroline Jane. "Development and characterization of an in vitro model for liver homeostasis." Thesis, University of Ottawa (Canada), 1994. http://hdl.handle.net/10393/6906.
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The major objective of this work was the development of an in vitro model for liver homeostasis which would allow the future study of early events in cell proliferation and cell death. The model which was set up involves growing T51B rat liver epithelial cells with a single dose of 1nM epidermal growth factor (EGF). This results in a period of hyperplasia where the cells reach double the control cell numbers two days after EGF addition. This is then followed by a decrease in cell numbers and the cell density returns to around the confluent control level five days after EGF addition. The model was investigated to ascertain whether the decrease in cell numbers three to five days after EGF addition was due to an increase in apoptosis. The results from light and electron microscopy studies, from the electrophoresis of T51B cell DNA and from the quantification of nuclear fragmentation indicated that the cells do die via an increase in apoptosis. The electron microscopy studies also show that healthy T51B cells can phagocytose apoptotic bodies. This suggests that the model is more physiological than other in vitro models of apoptosis. Cell growth studies and EGF binding studies were carried out in order to try to determine which events, if any, are EGF specific. The results from these studies suggest that occupancy of the low affinity binding site of the EGF receptor is responsible for the hyperproliferation seen when the T51B cells are grown with high doses of EGF. These studies also suggest that the apoptosis could be triggered by the down-regulation of the receptor, in a manner analogous to the removal of a trophic hormone in other systems. Thus this work describes the development and characterization of an in vitro model of liver homeostasis which closely parallels in vivo systems where animals are given mitogenic stimuli, and it also provides a good system for studying the role of EGF in cell proliferation and apoptosis.
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Douglas, O. "An evaluation of in vitro models for the assessment of mitochondrial toxicity within Drug Induced Liver Injury (DILI)." Thesis, University of Liverpool, 2017. http://livrepository.liverpool.ac.uk/3012327/.
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Drug Induced Liver Injury (DILI) is of major concern to both clinicians and the pharmaceutical industry due to the attrition of lead compounds during preclinical development and the incidence of hepatotoxicity and/or the withdrawal of drugs post marketing. It has been hypothesised that drug-induced mitochondrial dysfunction (DIMD) could be a causative factor of DILI. The nucleoside analogue fialuridine (FIAU) was withdrawn from use following a clinical trial in 1993 in which 5 patients died of hepatic failure. Subsequent research has shown that FIAU induces toxicity via mitochondrial DNA damage. However, current in vitro screening methods do not identify these compounds as toxic and thus this poses a problem to pre-clinical safety assessments. Three methods of metabolic modification, utilising glucose or galactose supplemented media and 2DG were compared to define and monitor mitochondrial toxicity induced by FIAU, and positive control adefovir (ADEF), in HepG2 (human hepatocellular carcinoma) cells. The structural isomer of fialuridine, (FIAU 2’ epimer) was included as a negative control. Mitochondrial toxicity could not be demonstrated in HepG2 cells over a 7-day period by any of these drugs. The HepaRG line is evident to be more hepatocyte-like than HepG2 cells and therefore can overcome the limitations of the widely used HepG2 cells. It is hypothesised that their increased primary human hepatocyte (PHH) like phenotypic characteristics may be more suitable for drug toxicity studies. Here, the utility of HepaRG based models in the detection of mitotoxicants was compared with previous research using a HepG2 model. Bioenergetic phenotyping revealed that the HepaRG line is less metabolically active when compared to HepG2 cells. HepaRG cells have the capacity to undergo metabolic modification using a short term glucose/galactose switch method and thus detect compounds with mitochondrial liabilities (EC50-ATPglu/EC50-ATPgal >2). Following the assessment of 12 hepatotoxins it was concluded that the HepaRG line offered no increased sensitivity for the detection of mitotoxicants compared with HepG2 cells. However, their stability in culture over extended periods (1 to 4 weeks) may be advantageous for the study of delayed toxicity. Therefore, HepaRG cells were further utilised in the assessment of FIAU induced mitochondrial toxicity, using an acute metabolic screen. Mitochondrial toxicity in the absence of cell death was demonstrated following 2-week drug incubation. The measurement of cellular respiration (using Seahorse technology) demonstrated a dose-dependent decrease in mitochondrial respiration in the absence of a decrease in mitochondrial mass. Further studies demonstrated significant drug-dependent decreases in the expression of nuclear encoded complex II, mitochondria-encoded complex IV, plus a decrease in mitochondrial DNA (mtDNA). The studies have demonstrated that the HepaRG cell model is a suitable model for the study of mitochondrial toxicity induced by nucleoside antivirals with evidence indicating that the mechanism of action via effects upon mitochondrial DNA matches the clinical mechanism of hepatotoxicity. Further studies utilising HepaRG were performed to assess the chemical and molecular pathways of toxicity induced by paracetamol. The studies provided evidence that HepaRG cells have the metabolic capacity for the turnover of parent compound to the toxic metabolite NAPQI. Furthermore, acute metabolic screening demonstrated that following short term incubations the parent compound paracetamol contains a mitochondrial liability in the absence of NAPQI-induced GSH depletion. The studies within this thesis have highlighted that the HepaRG line is suitable for the detection of mitotoxicants in which toxicity is delayed or which is mediated via CYP P450 catalysed-metabolites. Additionally, the studies provide much evidence as to the power of in vitro screening models in providing fine and detailed mechanistic information.
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Sams, Craig. "In vitro human liver metabolism of some industrial solvents and pesticides and incorporation of metabolic parameters into mathematical models." Thesis, University of Sheffield, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.434530.
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Essaouiba, Amal. "Development of a liver-pancreas in vitro model using microfluidic organ-on-chip technologies." Thesis, Compiègne, 2020. http://www.theses.fr/2020COMP2573.
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Le diabète mellitus, également désigné comme la maladie du siècle, est une pathologie mortelle qui affecte le système endocrinien. Les mécanismes liés à la rupture de la boucle de rétroaction, qui régule le métabolisme et induit le diabète, ne sont pas entièrement connus. La compréhension des mécanismes d'action de l'insuline est donc essentielle pour le développement de stratégies thérapeutiques efficaces afin du lutter contre cette maladie. Par conséquent, il est impératif de trouver un modèle robuste et fiable, capable de surmonter les limites de la culture cellulaire traditionnelle en 2D et de l'expérimentation animale, pour la recherche sur le diabète. L'objectif de cette thèse est de développer un nouveau modèle de co‐culture foie‐pancréas en utilisant des systèmes microphysiologiques avancés (MPs) afin d’aborder plus efficacement le mécanisme impliqué dans la régulation endocrinienne hépatique et pancréatique. Ce travail met en évidence la capacité des systèmes multi‐organes sur puce qui combinent la compartimentation avancée des cellules en 3D, la microfluidique et la technologie des cellules souches pluripotentes induites (iPSC), pour atteindre une complexité biologique élevée et des fonctions rarement reproduites par une seule de ces technologies d’ingénierie tissulaireDiabetes mellitus (DM) or the so called disease of the century is a life threatening dysfunction that affects the endocrine system. The mechanisms underlying the break in the feedback loop that regulates the metabolism and the consequent diabetes induction are not fully known. Understanding the mechanisms of insulin action is therefore crucial for the further development of effective therapeutic strategies to combat DM. Accordingly, it is imperative to find a robust and reliable model for diabetes research able to overcome the limitations of traditional 2D in vitro cell culture and animal experimentation. The aim of this thesis is to develop a new liver‐pancreas co‐culture model using advanced microphysiological systems (MPs) to tackle more effectively the mechanism involving the hepatic and pancreatic endocrine regulation. This work highlights the power of multi organ‐on‐chip systems that combines the advanced 3D‐cell compartmentalization, microfluidics and induced pluripotent stem cells (iPSC) technology to achieve a high biological complexity and functions that are rarely reproduced by only one of these tissue engineering technologies
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Boulais, Lilandra. "Cryogel-integrated hepatic cell culture microchips for liver tissue engineering." Thesis, Compiègne, 2020. http://www.theses.fr/2020COMP2561.
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L’un des enjeux de l’industrie pharmaceutique aujourd’hui est de développer des modèles de foie in vitro fidèles pour améliorer la prédictivité des études précliniques, notamment l’étude de la toxicité et de l’efficacité des médicaments candidats. Ces dernières années, l’ingénierie tissulaire, approche multidisciplinaire pour développer des tissus, a mené au développement de nouvelles méthodes de culture cellulaire. Parmi elles, les cultures de cellules en 3D ou en perfusion ont permis d’obtenir des activités hépatiques similaires à celles observées in vivo. L’objectif de cette thèse est de combiner ces deux méthodes de culture cellulaire pour créer un modèle de foie in vitro encore plus fidèle. Pour cela, nous cherchons à développer un cryogel d’alginate intégré en micropuce avec des propriétés mécaniques adaptables à celles du foie en fonction de l’état physiologique à reproduire (foie sain ou pathologique). Dans la première partie, nous développons et caractérisons le cryogel d’alginate au niveau microscopique et macroscopique, à l’extérieur (échantillons cylindriques) puis à l’intérieur de la biopuce. Trois paramètres sont étudiés ici : la température de cryopolymérisation, la concentration d’alginate ainsi que la quantité d’agents réticulants. Les propriétés mécaniques, la porosité, l’absorption, l’interconnectivité des pores et la résistance au flux sont analysés.La deuxième partie vise à cultiver des cellules hépatiques au sein de ce nouveau dispositif. Pour cette étude de faisabilité la lignée cellulaire HepG2/C3A est utilisée. Les résultats montrent des cellules viables et fonctionnelles (production d’albumine, transformation d’APAP). De plus, nous observons une structure tissulaire 3D, qui se maintient après retrait du cryogel d’alginate. La dernière partie a pour but de complexifier le modèle hépatique, notamment par des co-cultures. Pour se rapprocher de la structure du sinusoïde, des cellules hépatiques sont cultivées avec des cellules endothéliales (HUVEC) selon deux approches. De plus, la possibilité de suivre des cellules tumorales circulantes (MDA-MB-231) dans le système est étudiéeToday, one of the challenges for the pharmaceutical industry is to develop accurate in vitro liver models to improve the predictability of preclinical studies, in particular the study of the toxicity and efficacy of drug candidates. In recent years, tissue engineering, a multidisciplinary approach to develop tissues, has led to the development of new cell culture methods. Among them, cell cultures in 3D or in perfusion allowed to obtain hepatic activities similar to those observed in vivo. The objective of this thesis is to combine these two cell culture methods to create an even more accurate in vitro liver model. To do so, we are seeking to develop an alginate cryogel integrated into a microchip with mechanical properties adaptable to those of the liver depending on the physiological state to be reproduced (healthy or pathological liver).In the first part, we develop and characterize the alginate cryogel at the microscopic and macroscopic level, outside (cylindrical samples) and then inside the biochip. Three parameters are studied here: the cryopolymerization temperature, the alginate concentration and the quantity of cross-linking agents. Mechanical properties, porosity, absorption, pore interconnectivity and flow resistance are analyzed. The second part aims to culture liver cells within this new device. For this feasibility study the HepG2/C3A cell line is used. The results show viable and functional cells (albumin production, APAP transformation). In addition, we observe a 3D tissue structure, which is maintained after removal of the alginate cryogel. The last part aims to complexify the hepatic model, in particular by co-cultures. To get closer to the sinusoid structure, liver cells are cultured with endothelial cells (HUVEC) according to two approaches. In addition, the possibility to follow circulating tumor cells (MDA-MB-231) in the system is studied
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Chen, Liqiong. "The development of a novel in vitro model of human liver for the study of disease pathogenesis." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/11020/.
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The development of systems for the long term in vitro culture of functional liver tissue is a major research goal. The central limitation of experimental systems to date has been the early de-differentiation of primary hepatocytes in cultures. Several factors including cell-cell interaction, cell-matrix interaction, soluble factors and 3D structures have been identified as the keys to overcome this limitation. The first aim of this project is to compare the established 3D model, co-culture of hepatocytes and hepatic stellate cells (HSCs) on PDLLA coated surfaces, to other best available systems using collagen and Matrigel. The hypothesis is that hepatocytes functionalities, established by cell-cell interaction, 3D structures and soluble factors, can be further enhanced by introduction of cell-matrix interaction. In order to test the hypothesis, rat hepatocytes were cultured in five different systems, including monoculture of hepatocytes on collagen gel, in collagen-Matrigel sandwich, co-culture of hepatocytes and HSCs on collagen gel, in collagen-Matrigel sandwich and on PDLLA coated surface. Hepatocyte specific function assays, namely albumin secretion, urea secretion, testosterone metabolism by HPLC and CYP activities by LC-MS-MS, were used to analyze cell functionalities. Homo-spheroids were only formed in monoculture on collagen gel, but hetero-spheroids were developed in all the co-culture systems. The results of function assays showed hepatocytes in collagen-Matrigel sandwich configuration had the best secretion of albumin and urea and best CYP activities during the culture period. These data demonstrated the hypothesis that hepatocyte functions of the established model can be further improved by introduction of cell-matrix interaction. In addition to establishment of rat hepatocyte culture systems, hetero-spheroids of primary human hepatocytes and primary human HSCs on PDLLA coated plates were developed successfully, due to the great improvements of isolation and culture of primary human HSCs. However, hepatocyte function assays have not been applied yet. Hepatic cell lines have several advantages that are not applicable to primary cultured human hepatocytes, namely unlimited lifespan and stable phenotype. The immortalized Fa2N-4 cell lines have recently been assessed as replacements of primary human hepatocytes in CYP induction studies. The second aim of this study was to simultaneously characterize CYP1A2, CYP2C9, CYP3A4 and CYP2B6 induction in Fa2N-4 cells through assessment of mRNA, protein and activity endpoints for a range of prototypical compounds (previously assessed in human hepatocytes) with known positive and negative induction potential. LC-MS-MS and RT-PCR were used for assessment of activity and mRNA endpoints respectively. As a result, it is considered that Fa2N-4 cells offer a substitute for primary human hepatocytes for CYP1A2 and CYP3A4 induction but not for CYP2B6 due to lack of cytosolic CAR expression.
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Johann, Esther [Verfasser], Dieter [Akademischer Betreuer] Schrenk, and Stefan Otto [Akademischer Betreuer] Müller. "Improved early in vitro prediction of drug-induced liver injury in man: assessment of novel 3D hepatic models / Esther Johann ; Dieter Schrenk, Stefan Otto Müller." Kaiserslautern : Technische Universität Kaiserslautern, 2018. http://d-nb.info/1170154018/34.
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Danoy, Mathieu. "Development of a physiologically-relevant in-vitro microfluidic model for monitoring of pancreatic cancer cells interactions with the liver." Thesis, Lille 1, 2017. http://www.theses.fr/2017LIL10093/document.
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Le procédé de la métastase cancéreuse et sa compréhension sont devenus un des sujets majeurs de recherche en Biologie. En utilisant des modèles in-vitro en culture statique et dynamiques, nous avons étudié la possibilité de reproduire l’environnement physiologique in-vivo avec ces modèles. Nous avons développé un modèle de coculture hiérarchique dans des plaques à fond en PDMS. Composé d’hépatocytes, de pericytes et de cellules endothéliales. Dans différentes conditions, l’influence de ces cellules sur l’adhésion de cellules cancéreuses ou promyéloblastiques a été analysée ainsi que leur effet sur l’état inflammatoire du système. Afin de reproduire le flux sanguin et les forces de cisaillement présents in-vivo, le modèle a été transféré dans un système microfluidique. Le système se compose de trois canaux séparés par des micro-piliers, pouvant être remplis indépendamment. Les pericytes insérés dans du gel, les hépatocytes, les cellules endothéliales et finalement les cellules cancéreuses ont été injectés de façon successive afin de reproduire l’environnement in-vivo. Les cellules ont été trouvées viables durant toute la culture et des marqueurs relatifs au foie et à l’inflammation exprimés. L’influence des hépatocytes et des pericytes a été analysé. Il a été observé que les cellules cancéreuses adhérées dans le canal du haut étaient attirées par les autres cellules dans les diffèrent canaux. Les modèles établis posent de solides bases pour d’autres systèmes plus complexes et d’intérêt pouvant servir de complément aux modèles in-vivo lors de la recherche de nouvelles substances médicamenteusesThe cancer metastatic process and its understanding have been a major topic of interest for researchers in the past. Using in-vitro models in both standard culture conditions and in microfluidic devices, we investigated the feasibility of such models in the representation of the physiological in-vivo situation. We developed a hierarchical coculture model in PDMS plates, composed of hepatocytes, pericytes and endothelial cells. In different culture conditions, the influence of the different cells composing the model on the adhesion of cancer cells and promyeloblastic cells was investigated as well as the influence on the inflammatory state of the culture. To reproduce the in-vivo blood flow and shear stress to which the endothelial cells and the adhering cells are subjected, the model was then transferred into a microfluidic biochip. The device was composed of three channels, separated by micropillars and which could be filled independently one from another. Pericytes embedded in a hydrogel, hepatocytes, endothelial cells and finally pancreatic cancer cells could be inserted successively to reproduce the in-vivo hierarchical situation. Cells were found to viable after the culture and markers related to the liver and inflammation to be expressed. The influence of the presence of hepatocytes and pericytes was investigated by varying the culture conditions. It was found that pancreatic cancer cells were attracted by the cells in other channels in coculture. The established models lay the bases for more complex and relevant systems that could complement their in-vivo counterparts in the drug discovery process
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Lohr, Christiane [Verfasser], and Dieter [Akademischer Betreuer] Schrenk. "Effects of Polychlorinated Dibenzo-p-Dioxins, Polychlorinated Dibenzofurans, and Polychlorinated Biphenyls in Human Liver Cell Models (in vitro) and in Mice (in vivo) / Christiane Lohr. Betreuer: Dieter Schrenk." Kaiserslautern : Technische Universität Kaiserslautern, 2013. http://d-nb.info/1045604054/34.
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Euler, Henrik von. "Electrochemical treatment of tumours /." Uppsala : Dept. of Small Animal Clinical Sciences, Swedish Univ. of Agricultural Sciences ([Institutionen för kirurgi och medicin - smådjur], Sveriges lantbruksuniv.), 2002. http://epsilon.slu.se/v133.pdf.
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Llamas, Vidales Jose Ricardo. "Use of a 3D liver microreactor as an in vitro model for the study of bile acid synthesis and hepatobiliary circulation." Thesis, Massachusetts Institute of Technology, 2009. http://hdl.handle.net/1721.1/61226.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 149-165).
The liver regulates a myriad of vital functions including bile acid synthesis, hepatobiliary circulation, cholesterol homeostasis, drug metabolism, etc. This thesis focuses on the use of a 3D in vitro model of liver to study the effects of compounds and culture conditions on hepatobiliary transport and bile acid synthesis. In order to achieve this goal, protocols were developed to perform hepatic transport studies of bile acids in perfused 3D primary rat hepatocyte cultures. An established 2D sandwich culture model was used as a foundation for evaluation of variations in protocol parameters including culture medium composition and assay incubation times. In 2D sandwich cultures, dexamethasone (DEX) was essential for the formation of canalicular networks, whereas epidermal growth factor (EGF) disrupted the formation of these networks. Strikingly, EGF promotes cellular re-polarization and canalicular network formation in perfused 3D cultures, in contrast to its effect on 2D cultures. Perfused 3D cultures were found to have greater bile acid transport capabilities and closer to in vivo expression of certain liver transporters than 2D sandwich cultures on day 7. Perfused 3D cultures also had greater bile acid synthesis on day 7 than 2D cultures, however this was inhibited by EGF in both cultures. A numerical model was also developed for the reduction of experimental measurements necessary to determine pharmacokinetic parameters of hepatic transport, thus allowing for quantitative comparison of the effects of different culture conditions and culture platforms on transporter activity. The result of this thesis is the adaptation of a system for the study of hepatic transport and bile acid synthesis to 3D cultures. This system has also been previously shown to maintain other liver functions such as drug metabolism, and the work of this thesis thus allows for the concerted study of all these functions and potentially others.
by Jose Ricardo Llamas Vidales.
Ph.D.
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Ekins, Sean. "Maintenance and cryopreservation of xenobiotic metabolism in precision-cut liver slices : evaluation of an alternative in vitro model to isolated hepatocytes." Thesis, University of Aberdeen, 1996. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU543199.
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Enzyme-specific substrate activities were higher in Sprague Dawley rat hepatocytes than slices; testosterone (250M) hydroxylations (1.9-16.9 fold), 7-ethoxycoumarin (25M) (O-deethylation, 14.8 fold, glucuronidation, 3.1 fold), carbamazepine (50M) epoxidation (2.4 fold), styrene (2mM) diol formation (9.7 fold) and CDNB (50M)-glutathione conjugate formation (8.7 fold). Most importantly, the ratio of 7-hydroxycoumarin sulphate to glucuronide conjugation was higher in slices, in agreement with the lower rate of 7-hydroxycoumarin formation and indicative of slower diffusion of 7-ethoxycoumarin into slices. Metabolite formation was also higher in dog hepatocytes than slices, although these were thicker (436m), possibly accounting for the larger differences observed between the two in vitro models when compared with rat. In particular, CDNB-glutathione conjugate formation was 32 fold higher in hepatocytes than slices. Testosterone 6-hydroxylation in human liver was higher in isolated hepatocytes than in slices, although the differences were smaller (0.5-3 fold). 7-hydroxycoumarin glucuronidation was higher in human slices than in hepatocytes (3-4 fold), whilst 7-hydroxycoumarin formation was higher in hepatocytes than slices (2-12 fold and 6 fold, respectively). The differences between human samples could reflect the differing qualities of hepatocyte preparations. In the future, liver slices may be a means of reducing experimental animal use, whilst vitrification would allow continual availability of human liver slices for in vitro drug metabolism and toxicology studies.
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Shibany, Khaled Ayad S. "The development of an in vitro model using equine hepatocytes and liver microsomes for the study of drug metabolism in the horse." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/51310/.
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Horses are extensively exposed to various kinds of medication. However, limited information is available about how these medications are metabolised in this species. Hence, the development of strategies and methods that provide a better understanding of different metabolic pathways for different drugs is of major importance. Although, the horse is considered as a monogastric animal, it is also a hind gut fermenting animal, i.e. most of the feed is degraded in the cecum and colon. Thus, it is hypothesised that this could result in an evolutionary difference in xenobiotic metabolising enzymes between human and horses which may lead to different pharmacokinetics. Therefore, the main aim of this study is to develop an in vitro model as a preclinical testing system of newly developed substances for horses using freshly isolated, cryopreserved equine hepatocytes and liver microsomes. Fresh hepatocytes were isolated and liver microsomes were prepared using livers which were obtained from horses, post slaughter. Part of the freshly isolated hepatocytes were cryopreserved. A comparison metabolic study was carried out to compare the three in vitro systems. The intrinsic clearance (Clint) of three drugs used in equine medicine omeprazole, flunixin and phenylbutazone, was determined via the substrate depletion method. The determined Clint values were extrapolated to in vivo hepatic clearance (ClH) using two in vitro liver models, namely, well stirred and parallel tube models. To perform the extrapolation step, the values of microsomal protein per gram of liver (MPPGL) and the number of hepatocytes per gram of liver in the horse (HPGL), also known as the hepatocellularity, key scaling factors in both the well-stirred and parallel tube models, were obtained for horse. These scaling factors were determined by comparing the CYP P450 content in microsomes and cryopreserved hepatocytes against the CYP P450 content of the liver. Additionally, HPGL was also calculated from the ratio of liver protein concentration to matched hepatocyte suspension protein concentration. Effects of ageing on CYP P450 content, MPPGL and HPGL values were also investigated. Furthermore, chloramphenicol’s potential effect on the in vivo AUC for omeprazole was predicted using microsomes. In the present study, freshly isolated equine hepatocytes were successfully cryopreserved and the viability of recovered cells, after a 30% Percoll gradient, was 77 ± 11% and estimated recovery rate was approximately 27%. MPPGL and HPGL values ranged 41-73 mg/gram of liver (mean= 57 mg/gram of liver, n=39) and 146 - 320 × 106 cells/g of liver (Average = 227× 106 cells/g of liver, n=18) using CYP P450 method; and 156 - 352 × 106 cells/g of liver (Average = 232× 106 cells/g of liver) using protein method. It was found that increase in age had no effect on CYP P450/mg microsomal protein content. This study successfully predicted the in vivo clearance for omeprazole, flunixin and phenylbutazone using, both, freshly isolated and cryopreserved equine hepatocytes. Meanwhile, microsomes significantly underpredicted the in vivo hepatic clearance ClH. However, microsomes were used in present study to perform drug-drug interaction (DDI) in vitro study between omeprazole and chloramphenicol. The average of IC50 and the inhibitor constant Ki of the three horses were 17.7±5.8 μM and 15.4±5 μM, respectively. The work presented in this thesis paves the way to develop in vitro models using equine hepatocytes and liver microsomes, which are reproducible, scalable and compatible with screening platforms. These models can be applied to the improvement of predicted detection times (DT) in horse racing which may aid veterinarians to estimate accurate withdrawal times; hence, the horse welfare will be improved. Moreover, further phase II metabolism, drug induction and comparative studies can be performed.
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Cornu, Raphaël. "Nanoparticules et santé : de grandes promesses thérapeutiques, mais pour quel risque ?" Thesis, Bourgogne Franche-Comté, 2019. http://www.theses.fr/2019UBFCE018.
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Les nanoparticules sont définies comme des structures sphériques dont le diamètre maximum est de 100 nanomètres. Les domaines d’applications des nanoparticules incluant l’industrie agroalimentaire et pharmaceutique, sont extrêmement nombreux. L’Homme peut être quotidiennement exposé à des nanoparticules via différentes voies d’administration (orale, intraveineuse, pulmonaire et cutanée). En raison de leur taille nanométrique, les nanoparticules possèdent des propriétés physico-chimiques uniques, induisant de fortes interactions avec l’environnement biologique. Ces caractéristiques ont été largement exploitées pour la conception de nanomédicaments pour le diagnostic et la thérapie. Cependant, les problèmes liés à leur toxicité ont été mis en lumière parallèlement à leur développement. Ce travail de thèse vise à étudier la toxicité potentielle des nanoparticules. L'évaluation toxicologique a été réalisée à l'aide de modèles cellulaires adaptés aux voies systémique et orale. Les mécanismes impliqués dans la nanotoxicité ont été étudiés afin d’identifier des facteurs de toxicité. La première partie de ce travail a été consacrée à l’étude de la toxicité hépatique in vitro et in vivo des nanoparticules de PLGA et de silice. Les nanoparticules de PLGA sont utilisées comme vecteurs de médicaments alors que les nanoparticules de silice jouent le rôle d’agent antiagglomérant dans l’industrie alimentaire et également d’excipients pharmaceutiques. Leurs effets sur la fonction hépatique et en particulier sur l'activité des cytochromes P450 ont été évalués. La seconde partie de ce travail a consisté à étudier l’impact des nanoparticules de silice sur la barrière intestinale, en particulier sur la perméabilité paracellulaire et l’intégrité de la barrière. En mettant en exergue les différences interespèces ou le rôle protecteur du mucus, ce projet a démontré que le choix des outils toxicologiques était crucial pour une évaluation prédictive de la nanotoxicité. La taille, les propriétés de surface et la composition ont été identifiées comme des facteurs importants de toxicitéNanoparticles are defined as spherical structures with a maximum diameter of 100 nanometers. The application fields of nanoparticles including food and pharmaceutical industries are extremely broad. Human can be exposed daily to nanoparticles through various administration routes (oral, intravenous, pulmonary and cutaneous). Due to their size at the nanoscale, nanoparticles have unique physicochemical, inducing strong interactions with the biological environment. These features were widely exploited for the conception of nanomedicines for the diagnosis and the therapy. However, issues relative to their biological toxicity were addressed in the same time. This thesis work aims to investigate the potential toxicity of nanoparticles. Toxicological evaluation was performed using cell models adapted for the systemic and the oral routes. Mechanisms involved in the nanotoxicity were studied to identify toxicity factors. The first part of the work focused on the in vitro and in vivo hepatic toxicity of PLGA and silica nanoparticles. PLGA nanoparticles are used as drug carriers while silica nanoparticles play the role of anticaking agent in food industry and of pharmaceutical excipients. Their effects on the liver function and especially on the cytochrome P450 activity were investigated. The second part of the work consisted to study the impact of silica nanoparticles on the intestinal barrier, especially on the paracellular permeability and the integrity of the barrier. By emphasizing interspecies differences or the protective role of mucus, this project demonstrated that the choice of toxicological tools was crucial for a predictive nanotoxicity evaluation. Size, surface properties and composition were identified as major toxicity factors
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Georgiou, Leonidas. "DCE-MRI assessment of hepatic uptake and efflux of the contrast agent, gadoxetate, to monitor transporter-mediated processes and drug-drug interactions : in vitro and in vivo studies." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/dcemri-assessment-of-hepatic-uptake-and-efflux-of-the-contrast-agent-gadoxetate-to-monitor-transportermediated-processes-and-drugdrug-interactions-in-vitro-and-in-vivo-studies(d4b3bc62-8636-470b-90ae-38e25e7ee7be).html.
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Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) techniques offer the opportunity to understand the physiological processes involved in the distribution of the contrast agent in vivo. This work utilises a liver specific contrast agent (gadoxetate) and demonstrates the potential use of these techniques to study transporter-mediated process in vivo. In vitro experiments investigated gadoxetate’s interaction with uptake and efflux transporters at the cellular level, ideally a prerequisite to understand the contribution of transporter proteins in in vivo pharmacokinetics. MRI was used to measure the accumulation of gadoxetate in fresh rat hepatocytes. Furthermore, LC-MS/MS methodology was optimised in conjunction with two in vitro systems to determine the role of transporters in gadoxetate uptake and efflux. HEK-OATP1B1 transfected cells were used to optimise the LC-MS/MS technique and Caco-2 cell monolayers were used to examine whether gadoxetate is a substrate of the efflux transporters, Pgp and BCRP. Subsequent studies demonstrated the use of DCE-MRI techniques to study transporter-mediated processes. Two pharmacokinetic models were proposed to quantify the uptake and efflux of gadoxetate in vivo. The suitability of the models in describing the liver concentration profiles of gadoxetate was assessed in pre-clinical and clinical reproducibility studies. Further pre-clinical experiments demonstrated the ability of the proposed DCE-MRI techniques to monitor changes in the uptake and efflux rate estimates of gadoxetate into hepatocytes, through co-administration of the transporter inhibitor, rifampicin, at two doses. The work presented demonstrates the potential use of DCE-MRI techniques as a diagnostic probe to assess transporter-mediated processes and drug-drug interactions (DDIs) in vivo.
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Tian, Yinghua. "Liver and partial liver transplantation : new models and mechanisms of partial liver graft regeneration /." Zürich, 2005. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000253387.
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Orbach, Sophia Michelle. "Multi-Cellular Organotypic Liver Models for the Investigation of Chemical Toxicity and Liver Fibrosis." Diss., Virginia Tech, 2018. http://hdl.handle.net/10919/93313.
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The liver is responsible for lipid and glucose metabolism, protein and bile synthesis and the biotransformation of xenobiotics. These functions, performed by hepatocytes, are dependent on heterotypic interactions with other liver cell types and the stratified microarchitecture of the organ. In vitro liver models provide insights into the role of each cell type and perturbations upon external stimuli. Despite the dissimilarities to in vivo and rapid dedifferentiation, most liver studies utilize hepatocyte monocultures. These models lack heterotypic interactions causing inaccurate assessments of toxicity and disease. Only a limited number of 3D hepatic models incorporate the major liver cell types, and these cultures primarily focus on the hepatocyte response. We have developed 3D liver models that include all major hepatic cell types and recapitulate the layered architecture of the organ. These models maintain hepatic functions for up to four weeks and can be used to isolate the role and response of each cell type. We used these models to study two critical aspects of the organ -- acute hepatotoxicity and liver fibrosis.
There are tens of thousands of chemicals with undetermined effects on the human body. High concentrations of xenobiotics can cause acute liver damage and failure. Liver impairment can result in multiple organ failure, hepatic encephalopathy and death. Therefore, it becomes critically important to investigate hepatotoxicity in a time, cost and resource effective manner. Our 3D liver models were validated for hepatotoxicity testing with acetaminophen, a prototypic drug. We then adapted and optimized the models for high-throughput hepatotoxicity testing with automated procedures and primary human hepatic cells.
Liver fibrosis and cirrhosis are well-established consequences of chronic chemical exposure, infection and alcoholism. The initiating factors, end stages and resolution of fibrosis have been extensively studied. However, there is minimal information on the role of the local microenvironment in the progression of the disease from diseased to healthy tissue. We designed 3D liver cultures with a mechanical gradient to gradually model this transition through spatial and temporal perspectives. These findings demonstrate the versatility and accuracy of these 3D hepatic models in the investigation of liver toxicity and fibrosis.Ph. D.
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Jones, Elizabeth A. "Investigation and in vitro modelling of early liver development." Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.419699.
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Mbamalu, Godwin E. "In Vitro Modulation of Rat Liver Glyoxalase II Activity." Thesis, University of North Texas, 1988. https://digital.library.unt.edu/ark:/67531/metadc500992/.
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Glyoxylase II (Glo II, E.C. 3.1.2.6) catalyzes the hydrolysis of S-D-Lactoylglutathione (SLG) to D-Lactate and glutathione. This is the rate limiting step in the conversion of methylglyoxal to D-Lactate. The purpose of the present study was to determine whether or not a relationship exists between some naturally occuring metabolites and in vivo modulation of Glo II. We have observed a non-competitive inhibition (~ 45%) of Glo II in crude preparation of rat liver by GTP (0.3 mM). A factor (apparently protein),devoid of Glo II,when reconstituted with the purified Glo II, enhanced Glo II activity. This coordinate activation and inhibition of Glo II suggest a mechanism whereby SLG levels can be modulated in vivo.
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Lewis, Andrew L. "Flow cytometric analysis of hepatocyte proliferation in vitro." Thesis, University of Nottingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288990.
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Li, Wan-Chun. "In vitro transdifferentiation of liver into functional pancreatic-like cells." Thesis, University of Bath, 2006. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.425508.
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Tuckwell, Daniel S. "In vitro models for degenerative diseases." Thesis, University of Nottingham, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.277478.
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Thomson, Hazel. "Scale-up and 'in vitro' testing of the Strathclyde bioartificial liver." Thesis, University of Strathclyde, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.426362.
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Davies, Catherine Sarah. "In vitro assay of hydroxynaphthoquinones against the liver stages of 'Plasmodium'." Thesis, Imperial College London, 1989. http://hdl.handle.net/10044/1/47401.
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Ashfaq-Khan, Muhammad [Verfasser]. "Dietary wheat amylase trypsin inhibitors worsen chronic liver disease in preclinical models of non-alcoholic fatty liver disease and liver fibrosis / Muhammad Ashfaq-Khan." Mainz : Universitätsbibliothek Mainz, 2018. http://d-nb.info/1173844449/34.
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Hayward, Nicola Margaret. "Apoliprotein B metabolism in hamster livers, studied in vitro." Master's thesis, University of Cape Town, 1990. http://hdl.handle.net/11427/27176.
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This study aimed to investigate lipoprotein metabolism in male hamsters fed diets considered to be atherogenic in humans. Livers from adult male hamsters were selected to study aspects of apolipoprotein B metabolism. Isolated hepatocytes in suspension were compared with those maintained under tissue culture conditions. Liver slices were also prepared and compared with isolated suspended hepatocytes. Freshly prepared hepatocytes from the animals were incubated with radiolabelled precursors in suspension, or they were maintained under tissue culture conditions; liver slices were also investigated. The rates of total protein synthesis were of the same order in each of these systems, but protein secretion was impaired in liver slices, probably as a result of diffusion problems associated with the altered architecture of the sliced tissue. Albumin constituted 40 - 50% of the secreted proteins in each system. The rates of VLDL synthesis were increased in cells and slices prepared from animals previously fed sucrose- or fat-rich diets, but the secretion of VLDL was inhibited when diets contained unsaturated fat. The overall synthesis of apolipoprotein B was enhanced by fat-feeding; in the case of suspended hepatocytes, secretion of this protein was decreased when the preceding diet contained fats that were unsaturated; while in the case of liver slices, secretion was paradoxically enhanced. Apolipoprotein B was not degraded at significant rates in hepatocytes prepared from either control or fat-fed hamsters.
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Bruschi, Sam A. "Investigations into mechanisms of paracetamol-induced toxicity using ìn vitro' systems /." Title page, abstract and table of contents only, 1987. http://web4.library.adelaide.edu.au/theses/09PH/09phb192.pdf.
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Pathmanathan, Saidharshini. "Development of in vitro models of cerebral ischaemia." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.249162.
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Sivathanu, Vivek. "In vitro models for airway epithelial cell culture." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/81726.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2013.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 40-41).
This work is about the development of a physiologically relevant model of the human airway. Various factors such as the cell model, physiochemical factors such as the cell substrate properties including its stiffness, shear stress, stretch, the air-liquid interface and the biochemical factors in the medium influence the biology of the cells. The aim of this work is to closely approximate conditions in an in vivo situation by engineering the above conditions in to the in vitro platform. An assay to introduce the cell substrate properties was developed in a glass bottomed petri dish type culture as well as a microfluidic device culture. The influence of the cell substrate on airway epithelial cell monolayer formation was investigated in detail by changing the stiffness of the substrate independently by changing the gel concentration, the gel formation pH and the height of the gel from a hard substrate. Further, we found that biochemical growth factors have a huge role in cell monolayer formation. A real-time measurement of monolayer integrity using electrical resistance measurements was developed. A shear stress application platform was developed and a stretch application platform was designed. The applications of such a platform with the inclusion of various physiologically relevant factors include the study of physiologic evolution of microbes such as the influenza virus.
by Vivek Sivathanu.
S.M.
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Rhodes, Hannah Lucinda. "Genetic analysis and in vitro models of cystinuria." Thesis, University of Bristol, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.738194.
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Hurrell, Tracey. "Proteomic assessment of potential in vitro hepatotoxicity models." Thesis, University of Pretoria, 2016. http://hdl.handle.net/2263/59107.
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Scientifically credible and valid biological systems are essential in pharmaceutical research and development. Standardizing in vitro preclinical hepatotoxicity is confounded by the diversity of origin of cells and the ability to retain hepatocellular functions. Key determinants of valid hepatotoxicity models are resemblance to primary human hepatocytes (PHHs), adaptability to high-throughput screening and biological applicability. Numerous in vitro models, including immortalized cell lines and hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells (iPSCs), attempt to reflect features of PHH. Additional influencing factors are the mechanical and geometric environment which dictate functionality and suggest a role for spatial organization as a requirement for mimicking PHH. As there is poor correlation between the cellular genome and proteome, assessing the hepatic phenotypes using proteomics is essential to capture functional cellular responses. The aim of this research was to determine proteomic differences between PHHs and differentially cultured and sourced human hepatocyte-derived cell lines or differentiated HLCs. Additionally, hepatocyte models were used to generate non-specific, proteome-wide information associated with exposure to selected known hepatotoxins to identify potential proteomic signatures of hepatotoxicity.Thesis (PhD)--University of Pretoria, 2016.
Pharmacology
PhD
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Kalber, Tammy Louise. "In vivo susceptibility-contrast MRI studies of mouse models of liver metastasis." Thesis, St George's, University of London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.440446.
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Khademhosseini, Alireza. "In vitro study of bone marrow derived progenitor cells in liver-like microenvironments." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/MQ62916.pdf.
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Hammond, John Stotesbury. "Scaffolds for liver tissue engineering : in vitro co-culture & in vivo release." Thesis, University of Nottingham, 2009. http://eprints.nottingham.ac.uk/12556/.
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This thesis presents the development and evaluation of two applications for scaffolds in the field of liver tissue engineering. In the first study a poly (D,L lactic acid) (PDLLA) scaffold is used as a three-dimensional template for hepatocyte–hepatic stellate cell (HSC) co-culture. To enhance PDLLA ligand binding capacity scaffolds are surface modified using allylamine plasma deposition and treatment with NaOH. Primary adult rat hepatocytes and HSC are then seeded onto these scaffolds and cultured in static conditions. Scanning electron microscopy (SEM) is used to assess mono-culture and co-culture morphology whilst synthetic and cytochrome P450 function are measured using albumin and testosterone assays. The second study explores the potential for intrahepatic growth factor and extracellular matrix (ECM) delivery from a biodegradable polymer scaffold to promote liver growth and to enhance regeneration. The study is undertaken in rats. The scaffold design and implantation technique are first piloted in a short survival study. Hepatocyte growth factor (HGF), epidermal growth factor (EGF), fibroblast growth factor (FGF)1, FGF2 and liver derived ECM (L-ECM) are then loaded into poly(lactic-co-glycolic acid) (PLGA) + 5% poly(ethylene glycol) (PEG) scaffolds and implanted into normal and partially hepatectomised liver. Implant morphology is assessed by micro-CT reconstruction. Growth factor bioactivity and release are confirmed by in vitro profiling. Liver growth and volume redistribution are assessed by liver weight analysis. Parenchymal injury and function are quantified by measuring serum aspartate aminotransferase (AST) & bilirubin. 5-bromodeoxyuridine (BrdU) inclusion & MIB-5 immunohistochemistry (IHC) are used to identify hepatocyte and non-parenchymal cell proliferation. Liver-scaffold interaction is characterised by H&E and Masson’s trichrome staining. Non-parenchymal cell migration is assessed by ED-1 and desmin IHC. All histology is then subjected to image analysis.
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Kalezi, Artemis. "Tissue-engineered liver microreactor as an in vitro surrogate assay for gene delivery." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/38981.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2007.Includes bibliographical references.
The lack of correlation between in vitro and in vivo gene delivery experiments presents a significant obstacle in the progress of gene therapy studies by preventing the extrapolation of successful cell culture results into animals. This phenomenon has also been documented in the specific case of liver where standard hepatocyte culture systems fail to reliably predict the in vivo performance of gene delivery vectors. This is possibly a consequence of the loss of differentiated phenotype that these cells undergo when they are dissociated from their in vivo environment and cultured in vitro. This problem underscores the necessity for better in vitro models that can mimic the physiological environment and responses of in vivo liver tissue. This thesis aimed at developing an alternative in vitro gene delivery assay based on the Tissue-Engineered Liver Microreactor, a culture system designed to facilitate the morphogenesis of three-dimensional tissue-like structures from isolated liver cells under continuous perfusion, maintain cell viability and hepatic functionality for long-term culture periods and enable repeated in situ observation with microscopy. We developed experimental assays to non-invasively detect and quantify gene delivery efficiency in the 3D environment of the microreactor culture based on the application of 2-photon microscopy and spectroscopy.
(cont.) These techniques provide a convenient platform for comparative analysis of different vectors. Our main objective was to compare the gene delivery efficiency of an adenoviral vector (Ad5-CMV-EGFP) in the microreactor system and 2D hepatocyte monolayer culture. Quantitative assays were developed based on Real-Time PCR and RT-PCR to measure the levels of Ad vector uptake and transgene expression. The Ad mass transport in both systems was mathematically modeled to estimate the Ad uptake constant as a basis for comparison of delivery efficiency. This parameter was found to be significantly higher in the microreactor system, suggesting a more efficient mechanism of Ad internalization. Moreover, gene expression was measured in terms of transgene mRNA levels; the ratio of gene expression relative to Ad uptake was estimated as the basis for comparison of vector transcription efficiency. No significant difference was found between the 2 systems. These results provide some evidence that a more physiological culture system can yield different information (potentially more relevant to the in vivo situation) compared to standard in vitro culture.
by Artemis Kalezi.
Ph.D.
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Gunawardhana, Lhanoo 1959. "Human liver slices: An in vitro system for determination of N-acetylation and acetylator status." Thesis, The University of Arizona, 1989. http://hdl.handle.net/10150/291394.
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An in vitro system has been developed to study N-acetyltransferase (NAT) activity using human liver slices in dynamic organ culture. Acetylation of para-aminobenzoic acid (PABA) and sulfamethazine (SMZ) in the presence of human liver slices was monitored by measuring the disappearance of the parent amine from the incubation medium using the colorimetric procedure of Bratton & Marshall. Presence of the acetyl conjugate was confirmed using HPLC. PABA acetylation rates varied from 0.72-2.52 nmoles/hr/mg protein (n = 8). This small variation (4 fold) is consistent with the classification of PABA as a monomorphic substrate. The variation in the rate of SMZ acetylation was greater than 20 fold (0.144-3.68 nmoles/hr/mg protein; n = 9). This larger variation is characteristic of SMZ as a polymorphic substrate. The results obtained indicate that human liver slices in dynamic organ culture can be used for the determination of hepatic NAT activity and acetylator status of individual human livers.
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Larkin, Adam Lyston. "The Design of Three-Dimensional Multicellular Liver Models Using Detachable, Nanoscale Polyelectrolyte Multilayers." Diss., Virginia Tech, 2012. http://hdl.handle.net/10919/77190.
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We report the design and assembly of three-dimensional (3D) multi-cellular liver models comprised of primary rat hepatocytes, liver sinusoidal endothelial cells (LSECs), and Kupffer cells (KCs). LSECs and KCs in the liver model were separated from hepatocytes by a nanoscale, detachable, optically transparent chitosan and hyaluronic acid (HA) polyelectrolyte multilayer (PEM) film. The properties of the PEM were tuned to mimic the Space of Disse found in liver. The thickness of the detachable PEM was 650 to 1000 nm under hydrated conditions. The Young's modulus of the PEM was approximately 42 kPa, well within the range of modulus values reported for bulk liver. The 3D liver models comprised of all three cell types and a detachable PEM exhibited stable urea production and increased albumin secretion over a 12 day culture period. Additionally, the 3D liver model maintained the phenotype of both LSECs and KCs over the 12 day culture period, verified by CD32b and CD163 staining, respectively. Additionally, CYP1A1 enzyme activity increased significantly in the 3D liver models. The number of hepatocytes in the 3D liver model increased by approximately 60% on day 16 of culture compared to day 4 indicating. Furthermore, only the 3D hepatic model maintained cellular compositions virtually identical to those found in vivo. DNA microarray measurements were conducted on the hepatocyte fractions of the 3D liver mimic to obtain insights into hepatic processes. Gene sets up-regulated in the 3D liver model were related to proliferation, migration, and deposition of extracellular matrix, all functions observed in regenerating hepatocytes. Taken together, these results suggest that inter-cellular signaling between the different cell types in the 3D liver model led to increased hepatic functions. To the best of our knowledge, this is the first study where three of the major hepatic cell types have been incorporated into a model that closely mimics the structure of the sinusoid. These studies demonstrate that the multi-cellular liver models are physiologically relevant. Such models are very promising to conduct detailed investigations into hepatic inter-cellular signaling.Ph. D.
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Mahmood, Tahir. "Models of tissue bonding to bioactive glass in vitro." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape3/PQDD_0027/MQ50351.pdf.
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Chang, Cynthia J. "In vivo and in vitro models of distraction osteogenesis." Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.558214.
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Distraction osteogenesis (DO) is a unique process of bone formation used for clinical correction of skeletal deformities. In DO, bone is osteotomised, and the cut ends are slowly pulled apart by mechanical means to induce de novo ossification. Despite the extensive current and historical use of DO, the .mechanisms involved are not well understood. A novel mouse model of DO featuring a custom-developed external fixator was validated and characterised by using radiography, immunohistochemistry, and micro array techniques. The in vivo model was subsequently studied in a whole-transcriptome time course micro array analysis of DO. Genes relevant to osteogenesis, angiogenesis, mechanotransduction, cytoskeletal signalling, and the Wnt pathway were highly expressed. Time course statistical methods applied to the micro array data enabled profiling of global gene expression throughout DO and identification of genes and functions that showed significant differential expression over time. Concurrently, a novel three-dimensional in vitro model of DO was developed to assess the mechanobiological effects of distraction. The system consists of two pieces of hard mineral scaffold held in a rigid distractor. A cell-seeded fibrin clot bridges the scaffold ends to simulate the in vivo distraction gap. Using this in vitro model, the effects of a single application of tensile strain on the model were assessed. Digital image correlation demonstrated that strain patterns in the stretched construct are similar to those in the distraction gap. Additionally, murine osteoblasts were viable and proliferated in the scaffold-fibrin construct. Following a single distraction, cells exhibited elongated morphology, greater alignment, and increased alkaline phosphatase activity. In conclusion, a new mouse model and a novel in vitro model were shown to be useful correlates to clinical DO. Additionally, time course statistical analyses and in situ 3D staining techniques provide new tools for understanding and improving surgical bone lengthening. These findings serve as a promising starting point for future investigations into the key mediators of the regenerative potential unleashed in DO.
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Lomas, Emma C. "Investigation of post-mortem redistribution using in vitro models." Thesis, University of Huddersfield, 2018. http://eprints.hud.ac.uk/id/eprint/34635/.
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In unexplained death cases it is important to be able to determine the role (if any) of the drugs detected may have played in the death. However, drug concentrations can change between the time of death and the time of the analysis of the post-mortem sample, thus the concentration of the drug detected needs to be interpreted with caution. Post-mortem redistribution (PMR) is a process that involves the passive movement of drugs after death that can lead to changes in post-mortem drug concentrations at certain sites after death. In addition, other factors that could account for post-mortem changes include the environment in which an individual is found, as certain environments could accelerate decomposition, also the circumstances surrounding the death and the length of time between the death and recovery of the body. Certain organs including lungs, liver, and heart are depots of drugs for PMR as they can have higher concentrations than surrounding sites. The bladder has traditionally not been considered a possible depot for PMR. However one study, a case report, published in Japan discussing an individual that had a PMI of nine days with higher concentrations of diphenhydramine and dihydrocodeine in the femoral vein compared to the cardiac blood, has suggested that it may be. There have been no further studies to elucidate any possible role of the bladder in the PMR process. The aim of this thesis was to determine if the bladder is a potential site for PMR and to develop methodology to allow further study. The investigation included the influence of temperature, pH, porcine bladder degradation, and solution volume on diffusion from the bladder using in vitro diffusion through porcine bladder sections, whole porcine bladders and finally in vivo diffusion from the bladder in rat models over nine days. This thesis looked at three methods to investigate the possible diffusion of drugs from the bladder. 1) porcine bladder sections; 2) whole porcine bladders and 3) whole rats. The initial method used Franz Cells to determine the diffusion of rhodamine B, amitriptyline and amitriptyline’s metabolite nortriptyline across the porcine bladder wall. Acceptor chamber solutions were 20 mM pH 7.4 phosphate buffer (PBS) and 20 mM pH 5 ammonium acetate (AA). Donor solutions, dependent on experiment, contained 100 mg/L rhodamine B or amitriptyline and nortriptyline in the respective solutions. Sampling was over five days. Parameters included temperature (37 °C, 20 °C, and 5 °C), pH (7.4, 5), intra-variability of porcine bladder diffusion and tissue degradation. Quantitation methods of rhodamine B (UV, Agilent, Cary 60), amitriptyline, and nortriptyline (HPLC, Dionex Ultimate 3000) were validated according to SWGTOX guidelines. The femoral vein has been stated as the best site for sampling post-mortem blood and interpretation due to the isolation from the main viscera. However, due to the above case report suggesting redistribution from the bladder this is a possible factor that could affect this sampling site. The porcine bladder sections and whole porcine bladders were analysed to determine how much drug would diffuse through the tissue over the first 100 hrs after death, which is the initial steps in determining the likelihood of drugs diffusing from the bladder to the femoral vein. Whole porcine bladder studies used the validated UV method for rhodamine B. Experimental temperature was 20 °C. Full and half-filled porcine bladders contained rhodamine B (100 or 200 mg/L) dissolved in pH 7.4 PBS and pH 5 AA. Triplicate analysis performed using the UV spectrophotometer at 554 nm. The in-vivo study involved catheterizing a rat and inserting silver nitrate into the bladder then securing it for Computed Tomography (CT) analysis over nine days. There was increased diffusion of all three drugs at physiological temperature (37°C) with a peak rhodamine B concentration of 3.46 ± 2.72 mg/L (intra-bladder, pH 5), 6.69 mg/L and 6.69 ± 4.76 mg/L for amitriptyline and nortriptyline respectively (pH 7.4). The other parameters including solution pH and tissue degradation showed no significant difference for drugs diffusing through the bladder over 5 days. Concentration and volume was not a factor for rhodamine B diffusing through the whole porcine bladder tissue. There was an increase in drug diffusion over the five days with a peak concentration of 3.5 ± 1.02 mg/L (pH 7.4). The rat bladder was intact for two days, and then between 2-6 days, an opening was observed with leakage of solution. However, after day 7 this solution was not observed on the CT image. The CT data show that it is a good technique for the detection of diffusion of ions from the bladder, but would need to be further developed to look at the diffusion of larger molecular weight organic molecules. Based on this work, methods for investigating the diffusion of drugs across the bladder have been developed and validated. The use of μCT shows promise for the further visualization of PMR to investigate not only diffusion from the bladder but also diffusion from other drug depots in the body. However, based on this work it is unlikely that the bladder is a significant source of PMR to the femoral vein, at least in PMI of less than 100 hours after death.
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Williams, Kaylyn Renee. "In Vitro Models of Cellular Dedifferentiation for Regenerative Medicine." Thesis, Virginia Tech, 2018. http://hdl.handle.net/10919/83715.
Full textAbstract:
Stem cells have the ability to self-renew and to differentiate into a variety of cell types. Stem cells can be found naturally in the body, can be derived from the inner cell mass of blastocysts, or can be made by dedifferentiation of adult cells. Regenerative medicine aims to utilize the potential of stem cells to treat disease and injury. The ability to create stem cell lines from a patient's own tissues allows for transplantation without immunosuppressive therapy as well as patient-specific disease modeling and drug testing. The objective of this study was to use cellular dedifferentiation to create in vitro cell lines with which to study regenerative medicine. First, we used siRNA targeted against myogenin to induce the dedifferentiation of murine C2C12 myotubes into myoblasts. Timelapse photography, immunofluorescence, and western blot analysis support successful dedifferentiation into myoblasts. However, the inability to separate the myotubes and myoblasts prior to siRNA treatment confounded the results. This system has the potential to be used to study mechanisms behind muscle cell regeneration and wound healing, but a better method for separating out the myoblasts needs to be developed before this will be achievable. Second, we used a doxycycline-inducible lentiviral vector encoding the transcription factors Oct4, Sox2, cMyc, and Klf4 to create a line of naive-like porcine induced pluripotent stem cells (iPSCs). This reprogramming vector was verified first in murine cells, the system in which it was developed. Successful production of both murine and porcine iPSC lines was achieved. Both showed alkaline phosphatase activity, immunofluorescence for pluripotency marker (Oct4, Sox2, and Nanog) expression, PCR for upregulation of endogenous pluripotency factors (Oct4, Sox2, cMyc, Klf4, and Nanog), and the ability to form embryoid bodies that expressed markers of all three germ layers. Additionally, we were able to create secondary porcine iPSC lines by exposing cellular outgrowths from embryoid bodies to doxycycline to initiate more efficient production of porcine iPSCs. The secondary porcine iPSCs were similar to the primary porcine iPSCs in their morphology, behavior, alkaline phosphatase expression, and Nanog expression with immunofluorescence. The porcine iPSCs were dependent on doxycycline to maintain pluripotency, indicating that they are not fully reprogrammed. Despite this dependence on doxycyline, this system can be used in the future to study the process of reprogramming, to develop directed differentiation protocols, and to model diseases.Master of Science
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Cui, Yixiao. "Recapitulating Brain Tumor Microenvironment with In Vitro Engineered Models." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1595545538654859.
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Schjølberg, Tiril Helgesen. "In Vitro Synthesis of Metabolites of three Anabolic Androgenic Steroids, by Human Liver Microsomes." Thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for bioteknologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-22910.
Full textAbstract:
Anabolic androgenic steroids are substances frequently misused to improve physicalperformance in sports. To reveal substances misused as doping, athlete urinesamples are collected and tested. To identify the steroid and/or its metabolitesin urine, reference compounds must exist for comparison. The time-consumingand ethical concerns about in vivo excretion studies for the examination of thesecompounds, make the use of liver fragment microsomes an attractive alternative.The aim of this thesis was to synthesize and identify metabolites from known andrare anabolic androgenic steroids, by the use of human liver microsomes. Liveris an important organ in steroid metabolism. By incubating AAS with humanliver microsomes and co factors, an in vitro simulation of the liver metabolism wascarried out. A conrmation of metabolites was performed by gas chromatographytandem mass spectrometry in full scan, MRM mode, or both. 6beta-hydroxymethylmetandienon, epimetandienon and 17,17-dimethylmetandienon were successfullysynthesized from metandienon, and the 17beta-hydroxymetandienon was producedfrom the 17,17-dimethyl metabolite. Respectively three and one metabolite(s)were found for the "designer steroids" methylnortestosteron and madol. Metabolitevariations were observed regarding the optimal time of incubation, and enzymaticrequirements of formation.
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Wallace, Karen. "Glucocorticoid-mediated trans-differentiation of the pancreas to liver, in vitro and in vivo." Thesis, University of Newcastle Upon Tyne, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.512022.
Full textAbstract:
The AR42J B-13 (B-13) rat acinar cell line trans-differentiates into functional hepatocyte-like cells (B-13/H) in response to glucocorticoid without the need for any complex extracellular matrix or treatment with expensive recombinant proteins. B-13 cells expressed pluripotency markers but did not respond to factors associated with the embryonic development of the liver. B-13 cells were readily expanded in culture and could provide a potentially unlimited supply of quantitatively functional hepatocyte-like cells. The role of the WNT signalling pathway - specifically the activity of the proximal WNT regulated TCF/LEF transcription factors - was examined. Trans-differentiation was dependent on a transient suppression of TCF/LEF transcriptional activity leading to the expression of the liver-enriched transcription factors C/EBPβ and proteins. Trans-differentiation was associated with a decrease in total β-catenin and increased phospho-β-catenin expression; whilst recruitment to the plasma membrane, cytosolic degradation and nuclear exclusion of β-catenin was a hallmark of B-13/H cells. Silencing of β-catenin induced trans-differentiation and repression of TCF/LEF signalling in the absence of glucocorticoid whilst glucocorticoid mediated trans-differentiation was abrogated when mutant forms of β-catenin were over expressed or cells were pre-treated with a WNT agonist. Analysis of the in vivo response to glucocorticoid using an adult rat model, a transgenic mouse model of Cushing's disease and patients treated with glucocorticoid was made. Results showed that the B-13 cell response to glucocorticoid is physiologically relevant and related to a normal patho-physiological response of mature pancreatic acinar cells to elevated levels of circulatory glucocorticoid. Data suggest that the expression of liver genes in pancreatic acinar cells in vivo is the result of direct trans-differentiation of mature acinar cells.