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1
Li, Daqing. "Entorhino-hippocampal projections in organotypic cultures." Thesis, University College London (University of London), 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315340.
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Mbobo, Buchule. "Modelling neuroimmune interactions using organotypic slice cultures." Master's thesis, University of Cape Town, 2017. http://hdl.handle.net/11427/24907.
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Tuberculosis predominantly manifests in the form of a pulmonary infection, but may spread out into other parts of the body and is then referred to as extrapulmonary tuberculosis (EPTB). One form of EPTB is an infection of the central nervous system (brain & spinal cord), CNS-TB. Although CNS-TB is relatively rare, accounting for about 5% of EPTB, it is characterised by high morbidity and mortality, particularly for children and immunosuppressed individuals. To examine the effects of a Mycobacterium tuberculosis infection of neural tissue, researchers have hitherto relied on two animal models namely, in vivo intracranial infections or in vitro culturing with dissociated neural cells. Both models have yielded crucial insights concerning CNS-TB but each have limitations e.g. lack of access to the brain during infection in vivo and absence of the 3D organizational tissue structure in vitro. This study investigated the effect of the vaccine strain for tuberculosis, Bacille Calmette-Guerin (BCG) on neural tissue using the model of organotypic hippocampal slice cultures; an in vitro model that overcomes the previously mentioned obstacles. The study sought to expound on immunological and electrophysiological responses to the infection. A viable and moderate BCG infection was established in the hippocampal slice cultures, confirmed by colony forming units enumeration and immunohistochemistry. However, immunological analysis using ELISA found that BCG infection did not change the production levels of cytokines and elicit a distinguishable immune response. To examine the neuronal function during BCG infection, whole-cell patch clamp technique was applied to the hippocampal slice cultures. The neuronal intrinsic properties were not significantly different between infected and non-infected slices. However, tuberculin PPD (M. tuberculosis extract) moderately and transiently had a depolarizing effect when 'puffed' directly onto neurons. In conclusion, organotypic slice cultures are suitable for the investigation of cellular interactions and neural functions in CNS-TB, and the neuronal impact of PPD warrants further investigation.
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Steinmeyer, Joseph D. (Joseph Daly). "Rapid single-cell electroporation for labeling organotypic cultures." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/60188.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2010.<br>Vita.<br>Includes bibliographical references (p. 37-39).<br>Single-cell electroporation is a technique for transfecting individual cells in tissue culture at relatively high efficiencies, however it is both time-consuming and low-throughput and this limits the number of different labeling agents that can be effectively introduced into a region of tissue in reasonable periods of time. A novel system that will rapidly load, clean, and accurately position a glass micropipette electrode into tissue culture for single-cell electroporation is proposed. The system will significantly increase the number of different labeling agents that can be introduced into a single tissue culture per unit time. This in turn, will provide a means for improving the study of neural anatomy at cellular resolutions in both tissue culture and in vivo environments.<br>Supported by grants from the National Institutes of Health and by the MIT Dept. of Electrical Engineering and Computer Science<br>by Joseph D. Steinmeyer.<br>S.M.
4
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.<br>Ph. D.
5
Kazmi, B. "Oral and dermal fibroblasts in 3D organotypic co-culture." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/17485/.
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It is widely observed that dermal wound healing results in the re-establishment of skin continuity through the formation of a scar. In comparison oral wounds heal more rapidly, with less visible scarring. The diverse nature of sub-epithelial fibroblasts has targeted them as mediators for such a distinction in phenotype. Fibroblasts and their differentiated form, the myofibroblast (characterised by the expression of α-SMA), are chief synthesisers of the ECM during wound healing. Myofibroblasts in particular play an important role in wound contracture, ECM synthesis and remodelling during normal wound healing and scar production; but are also observed in fibrosis and pathological scarring. The most potent mediator of this phenotype in the cytokine TGFβ1, which is abundantly present throughout wound healing. Here we used organotypic co-cultures and mono-cultures to assess the differences in phenotype and the relative contribution of topographically distinct fibroblasts within them. There is currently little data on the responsiveness of oral buccal mucosal and skin fibroblasts to TGFβ1, particularly when keratinocytes are present in a 3D environment. Here, for the first time, we used heterotypic organotypic co-cultures containing skin and oral buccal mucosal fibroblasts and added TGFβ1 under both resting and tethered conditions. We monitored the changes in response of these fibroblasts with and without the presence of keratinocytes, and subsequently assessed changes in phenotype upon substitution of the dermal fibroblasts with those from the reduced scarring oral buccal mucosa. Under resting conditions oral fibroblast seeded organotypics showed a lower constitutive myofibroblast expression, and were unresponsive to TGFβ1 mediated α- SMA expression, regardless of substrate compliance. These fibroblasts were found to significantly promote epithelial maturation under resting conditions, and express higher levels of active MMP-9 (and enzyme involved with keratinocyte migration) upon treatment with TGFβ1, when compared to dermal fibroblast seeded counterparts. When oral fibroblasts were introduced into a dermal organotypic co-culture environment, we observed a preferential change α-SMA phenotype, offering the potential for use of these cells in an area distinct from there origin to a favourable effect.
6
Frantseva, Marina. "H¦2O¦2 induced toxicity in rat organotypic hippocampal cultures." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape11/PQDD_0028/MQ51596.pdf.
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Phillips, Wiktor Samuel. "Studies of Respiratory Rhythm Generation Maintained in Organotypic Slice Cultures." W&M ScholarWorks, 2016. https://scholarworks.wm.edu/etd/1499449864.
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Breathing is an important rhythmic motor behavior whose underlying neural mechanisms can be studied in vitro. The study of breathing rhythms in vitro has depended upon reduced preparations of the brainstem that both retain respiratory-active neuronal populations and spontaneously generate respiratory-related motor output from cranial and spinal motor nerves. Brainstem-spinal cord en bloc preparations and transverse medullary slices of the brainstem have greatly improved the ability of researchers to experimentally access and thus characterize interneurons important in respiratory rhythmogenesis. These existing in vitro preparations are, however, not without their limitations. For example, the window of time within which experiments may be conducted is limited to several hours. Moreover, these preparations are poorly suited for studying subcellular ion channel distributions and synaptic integration in dendrites of rhythmically active respiratory interneurons because of tortuous tissue properties in slices and en bloc, which limits imaging approaches. Therefore, there is a need for an alternative experimental approach. Acute transverse slices of the medulla containing the preBötzinger complex (preBötC) have been exploited for the last 25 years as a model to study the neural basis of inspiratory rhythm generation. Here we transduce such preparations into a novel organotypic slice culture that retains bilaterally synchronized rhythmic activity for up to four weeks in vitro. Properties of this culture model of inspiratory rhythm are compared to analogous acute slice preparations and the rhythm is confirmed to be generated by neurons with similar electrophysiological and pharmacological properties. The improved optical environment of the cultured brain tissue permits detailed quantitative calcium imaging experiments, which are subsequently used to examine the subcellular distribution of a transient potassium current, IA, in rhythmically active preBötC interneurons. IA is found on the dendrites of these rhythmically active neurons, where it influences the electrotonic properties of dendrites and has the ability to counteract depolarizing inputs, such as post-synaptic excitatory potentials, that are temporally sparse in their occurrence (i.e., do not summate). These results suggest that excitatory input can be transiently inhibited by IA prior to its steady-state inactivation, which would occur as temporally and spatially summating synaptic inputs cause persistent depolarization. Thus, rhythmically active interneurons are equipped to appropriately integrate the activity state of the inspiratory network, inhibiting spurious inputs and yet yielding to synaptic inputs that summate, which thus coordinates the orderly recruitment of network constituents for rhythmic inspiratory bursts. In sum, the work presented here demonstrates the viability and potential usefulness of a new experimental model of respiratory rhythm generation, and further leverages its advantages to answer questions about dendritic synaptic integration that could not previously be addressed in the acute slice models of respiration. We argue that this new organotypic slice culture will have widespread applicability in studies of respiratory rhythm generation.
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Jussila, T. (Tommi). "Modelling cancer: recapitulation of tumor growth in experimental systems in vivo and in vitro." Doctoral thesis, University of Oulu, 2000. http://urn.fi/urn:isbn:9514256433.
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Abstract
The purpose of the study was to evaluate model systems of cancer development and
compare some of their critical features with cancer development in
vivo. Ovarian and endometrial cancers in man were used as correlates.
Tumor development in experimental animals, exposed to carcinogens and UV
irradiation, showed the entire spectrum of tumor development as compared to
spontaneous carcinomas: hyperplasia, dysplasia, benign papillomas and malignant
squamous cell carcinomas. For short-term analysis of differentiated homogenous
cell populations, the transplant model proved most useful. For long term
analysis of effects of extraneous agents, the skin carcinogenesis model is
probably the most rewarding.
Analysis of proliferation markers in human tumor samples as studied by
immunohistochemistry, showed that an increased expression of PCNA and Ki-67 was
associated with poor prognosis in ovarian neoplasms. Analysis of cell
proliferation in model tumors showed that the transplant model has a better
sensitivity when compared to the animal skin model and the subcutaneous
injection model, in that effect of changes in cell-host interaction on the
location and extent of the proliferating cell population can be studied therein.
The expression of some growth factors, their receptors, oncogenes and suppressor
genes were studied in ovarian and endometrial carcinomas and in skin cancer
model system in mouse exposed to carcinogens and UV irradiation. Variability in
expression and methodological problems precluded detailed analysis of these
markers in different models.
The expression of TGFβ1, TGFβ2 and TGFβ3 was determined in normal
human keratinocytes, and in 7 immortalized and
ras-transfected benign and malignant keratinocyte cell
lines, maintained as transplants and as subcutaneous tumors in nude mice. By
differential immunohistochemical localization of TGFβ isoforms, we
demonstrated that each isoform may serve specific roles in tumor development and
progression. The complex nature of TGFβ expression prevented detailed
analysis of isozymes in different models, the results in this study, however,
indicated a similar pattern in the models analyzed.
Morphological methods were used to determine relationship between epithelial
growth and formation and deposition of collagens in the extracellular matrix in
experimental models and human tumors. The composition of the mesenchyme differed
in tumors originating from different cell lines reflecting functional
interaction between epithelial cells and the mesenchyme in neoplastic
development. Tumor-stroma interaction was distinct in human, comparable
alterations were observed in experimental models, more so in transplants, less
in subcutaneous tumors, affecting tumor growth and differentiation in the
different models.
9
Acheva, Anna Rumenova. "Mechanisms of response to targeted irradiation in organotypic 3D skin cultures." Thesis, Queen's University Belfast, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.579565.
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Low linear energy transfer (LET) ionizing radiation as gamma and X-rays is widely used in the modem medicine for diagnostic and treatment purposes. Despite all the advantages that it gives for the early diagnosis and in the radiotherapy treatment of cancer, there are still unclear aspects about the mechanisms of the radiation effects in human tissue. Of particular interest is the detailed pathway which the directly irradiated cells use to communicate to their neighbours and its possible implications for radiotherapy applications. The aim of this project is to study the spatio-temporal signalling from irradiated to non- irradiated cells using in vitro 3D tissue models. For evaluation of radiation induced effects we used conventional 225 kVp X-ray and lead shielding to observe the effects in the non-targeted cells. Additionally we applied the novel 30 kVp micro collimator that could irradiate samples in wide 1-10 urn lines. The application of this targeted radiation source together with the shielding irradiation set up will cast light on the effects induced by localized radiation exposures and the signalling from the irradiated to non-irradiated cells using 3D organotypic skin as a model. Due to its fast cell turnover, the human epidermis is extremely sensitive to IR. This has a limiting effect on the radiotherapy as severe normal skin responses can delay treatment and even decline patients from radiotherapy. The current work is aiming to reveal the mechanisms of DNA damage, repair and their later consequences for the differentiation and development of inflammatory-like response in the irradiated and surrounding areas within the 3D organotypic skin model. We investigated use of inhibitors of pro-inflammatory pathways such as the transcription factor NF-KB and its downstream target COX-2, in order to reduce the signal transduction from the irradiated to non irradiared cells and to reduce the inflammatory responses in the surrounding normal tissue. Similar methods of control of the signal diffusion could be applied in the radiotherapy to reduce the inflammatory skin responses and decrease the range of the late side effects developing from the chronic inflammation that could further lead to ulceration and skin necrosis.
10
Froeling, Fieke E. M. "Tumour–stroma interactions in an organotypic culture model of pancreatic cancer." Thesis, Queen Mary, University of London, 2011. http://qmro.qmul.ac.uk/xmlui/handle/123456789/705.
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Pancreatic cancer is characterised by an intense fibrotic, or desmoplastic, stroma, which contributes to tumour progression. Three-dimensional in vitro culture models incorporating this non-tumour component may more closely recapitulate the complex in vivo situation. The aim of my project was to develop a physiologically relevant, three-dimensional organotypic culture model of pancreatic cancer to study the tumour-stroma interactions and its modulation by novel therapeutic agents. Cancer cells cultured on top of collagen/Matrigel gels, embedded with or without stromal cells (hTERT immortalised PS1 stellate cells or MRC5 fibroblasts), differentiated into luminal structures, exhibiting a central apoptotic core with a proliferating peripheral rim and apicobasal polarity. Stromal cells induced a reduction in total tumour cell number, which was associated with a decrease in E-cadherin expression, upregulated β-catenin expression and translocation of ezrin from the apical to the basal aspect of cancer cells, where it was associated with invasive activity. Subsequently, this organotypic model was raised to an air-liquid interface to study the direct and indirect effects of all-trans retinoic acid (ATRA), which rendered stellate cells back to their quiescent phenotype. Indirect effects of quiescent stellate cells on pancreatic cancer cells included changes in proliferation (decrease), apoptosis (increase), invasion (decrease), Wnt/β-catenin signalling (decrease) and an altered morphology. The Wnt/β-catenin signalling 6 perturbations were mediated by restoration of sFRP4 (secreted frizzled-related protein 4) secretion by quiescent stellate cells, resulting in reduced cancer cell invasion (reporter and invasion assays). All such observations could be validated in human pancreatic cancer tissue samples. Taken together, pancreatic organotypic culture offers a reproducible, in vitro three-dimensional culture model, which allows the study of tumour-stroma interactions in a physiologically relevant system. For treatment of pancreatic cancer, a tumour characterised by a poor response to conventional chemotherapeutic drugs, targeting the tumour-stroma cross-talk with agents such as ATRA offers an exciting novel therapeutic strategy.
11
Fell, Benjamin. "Organotypic human skin disease models for the assessment of novel therapeutic approaches." Thesis, Queen Mary, University of London, 2017. http://qmro.qmul.ac.uk/xmlui/handle/123456789/24594.
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Comprehensive in vitro modelling of inflammatory human skin conditions is an essential first step in the development and assessment of potential therapeutic approaches. Mouse models or monolayer keratinocyte cultures come with distinct limitations which might be complimented or overcome by the use of human-specific organotypic 3D culture models. Over the course of this thesis, an organotypic culture system, based on patientderived immortalised keratinocyte cell lines on a dermal equivalent collagen 1 gel, was established and used to recapitulate phenotypical features for two hereditary skin diseases, Harlequin ichthyosis and Tylosis with oesophageal cancer. Small molecular compounds, supplied via the medium, or RNA interference were used to modulate disease-specific changes in histology and marker expression of the skin equivalent. Since hyperproliferative skin conditions can be associated with an aberrant wound healing phenotype, the organotypic system was manipulated to obtain a basic in vitro wound healing model. This model displays typical features of re-epithelialisation over time (both normal and disease-specific) which can further be manipulated via shRNAmediated knockdown or the exogenous supply of compounds. In parallel, a non-disease model was used to assess the topical application of novel nanopolymeric drug delivery systems in regard to their ability to penetrate across the permeability barrier. Penetrance profiles for the organotypic model (in dependence of co-application with chemical enhancers) showed a similar pattern as for topical applications performed in parallel on explant skin. In conclusion, a highly adaptable human organotypic keratinocyte culture model was developed and used to recapitulate (and manipulate) skin disease phenotypes and epidermal wound healing in vitro, as well as perform first essential assessments of novel drug delivery systems.
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Rioult-Pedotti, Marc Guy. "Optical multisite recording of neural activity patterns in organotypic spinal cord tissue cultures /." [S.l.] : [s.n.], 1991. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=9393.
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Ajani, Gati. "Characterization of changes in hyaluronan following epidermal barrier injury in an organotypic model." Cleveland, Ohio : Cleveland State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=csu1209996841.
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Thesis (D.Eng.)--Cleveland State University, 2008.<br>Abstract. Title from PDF t.p. (viewed on July 7, 2008). Includes bibliographical references (p. 150-176). Available online via the OhioLINK ETD Center. Also available in print.
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El-Tarhouni, Amal Ibrahium. "Studies on the mechanosensory innervation of muscle using organotypic culture, reinnervation and immunohistochemistry." Thesis, Durham University, 1996. http://etheses.dur.ac.uk/5266/.
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This thesis studies sensory innervation in mammals using an organotypic co-culture of spinal cord-dorsal root ganglion and skeletal muscle of embryonic rat, the histological changes of reinnervated muscle spindles after nerve section and the localisation of the calcium-binding protein calretinin in cat mechanoreceptor organs. The immediate importance of this project concerns the better understanding of how the normal process of development differs from reinnervation following nerve lesion or section. A range of classical and well defined materials and methods as been used in the work described. The thesis Is divided into Ove chapters: Chapter 1 reviews aspects of the mechanosensory organs which have been studied experimentally in relation to their sensory innervation, including proprioceptive muscle spindle development, reinnervation, and finally, the presence of the calcium-binding protein, calretinin in the mechanoreceptor organs. This provides an introduction and background to the work. Chapter 2 describes the organotypic organisation of spinal-cord, dorsal-root ganglia and skeletal muscle co-culture in vitro. Results show that slices of the spinal-cord, dorsal- root ganglia survive well under experimental conditions and can live for several weeks with feeding every 1-3 days. Sensory neurons can develop and grow in a medium without any additional promoting factor. The presence of structurally identifiable synapses indicates that other neurons are also maintained in culture and have functional connections. In the organotypic culture new muscle fibres can form either from the original explant or from the additional explant. In chapter 3 I describe two abnormal endings present in spindles of the tenuissimus of the cat that had been reinnervated following section of the nerve more than one year previously. The reconstruction of the endings of these two spindles supports the hypothesis of modulation of the primary-ending response by the mechanical properties of the intrafusal muscle fibres, rather than by intrinsic properties of the la afferent itself. They further indicate that, in the absence of a la afferent, intrafusal-fibre differentiation can be maintained by a group II afferent. Chapter 4 concerns the localisation of the calcium-binding protein calretinin, which was studied immunohistochemically in the abductor digiti quinti medius muscle of the cat hind limb. The calretinin immunoreactivity was found in some intrafusal fibres, the primary endings and the cqjsule of the muscle spindles and the sensory terminals of tendon organs and Paciniform corpuscles. The present findings contradict a recent hypothesis that calretinin is associated with rapid adaptation, but suggest that calretinin has a specific function in muscle proprioceptors. Finally, Chapter 5 outlines the conclusions of this study and gives some suggestions for continuation of the work in the future.
15
Gronbach, Leonie [Verfasser]. "Organotypic head and neck cancer models for advanced preclinical drug testing / Leonie Gronbach." Berlin : Freie Universität Berlin, 2021. http://d-nb.info/1230407316/34.
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Medelin, Manuela. "Degenerative processes in organotypic spinal slices: challenging pre-motor network with stress conditions." Doctoral thesis, Università degli studi di Trieste, 2014. http://hdl.handle.net/10077/10130.
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2012/2013<br>My PhD project concerns neurodegenerative processes in the mouse spinal cord, with a particular attention to amyotrophic lateral sclerosis (ALS). During my PhD I have used as a model the organotypic spinal slice cultures and I have focused my studies on: early changes in spinal tissue excitability in an ALS genetic model; spinal network activity changes induced by oxidative stress in wild type (WT) and synaptic activity in premotor circuits when challenged by neuroinflammation in WT. The principal aim of my work was to understand the dialogue between a general stress condition and the spinal premotor network. To this aim, I combined electrophysiological techniques and immunofluorescence analysis to characterized the ventral interneurones located in spinal microcircuits. For that purpose I exploited the organotypic cultures developed from embryonic mouse spinal cord that are generally accepted as a good model to study the neuronal premotor activity and provide high experimental access to interneurones (Avossa et al., 2003).
In the first part of my research I compared WT cultures with SOD1G93A transgenic cultures, one of the more investigated ALS model. In cultured spinal networks, as described in acute preparation collected at different stages of development, there is a progressive fastening of glycinergic currents, represented by the reduction of the decay time constant (tau value) of synaptic currents along with the slices growth. WT and SOD1G93A cultures display a different maturation profile since in transgenic slices this developmental process is significantly steeper not only in glycinergic post synaptic currents (PSCs) but also in miniature PSCs (mPSCs). This difference in the glycinergic PSCs kinetic properties can be strongly reduced by the presence of TBOA that lowers the GABA synthesis. These results support the hypothesis that in SOD1G93A cultures there is an increase amount of glycine and GABA co-release leading to the conclusion that the synaptic release is conditioned by the presence of the mutation at an early stage of development, before any evident neuronal degeneration. Moreover, I supported this data also with preliminary results regarding the co-staining of GlyT2 and GAD65 (markers for presynaptic glycine and GABA, respectively). In fact, SOD1G93A spinal organotypic slices seem to display an higher amount of mixed synapses.
Next, I tested other stress processes of the tissue that could potentially affect synaptic activity and, ultimately, alter network activity. I tested chronic incubations of the spinal slices, since they are long-term preparations, with stress key players: hydrogen peroxide (H2O2) to create an oxidative stress and lipopolysaccharide (LPS) or a mixture of cytokines (CKs: TNF-α, IL-1β and GM-CSF) to mimic neuroinflammation. For these sets of experiments I have used another strain of mice with no genetic manipulation. All these chronic treatments increase the AMPA receptor mediated PSCs frequency; moreover, a neuroinflammation state is able to enhance the overall network activity; LPS treatment increases also the amplitude of AMPA-mediated synaptic currents in both spontaneous and miniature events, while CKs accelerate the disinhibited burst rhythm induced by the pharmacological removal of the synaptic inhibition that switch on the rhythmogenic centre contained in the spinal network. Summarizing, I detected that the treatments with these environmental cues affected the synaptic component, in this case the excitatory one, of the premotor network.
Altogether, my work highlighted that a genetic predisposition (in the case of familial ALS) and environmental factors of different kind (oxidative and inflammatory factors or an alterate SOD1) trigger changes in synaptic transmission and we may speculate that these alterations in the premotor circuit could cooperate in synergy leading to the development of misconnected networks that contribute to induce motoneuronal neurodegeneration.
Riassunto
Il mio progetto di dottorato riguarda processi neurodegenerativi del midollo spinale, con una particolare attenzione verso la sclerosi laterale amiotrofica (SLA). Durante il mio dottorato ho usato come modello le fettine organotipiche di midollo spinale e ho concentrato i miei studi su: cambiamenti precoci nell’eccitabilità del tessuto spinale in un modello genetico di SLA; cambiamenti nell’attività del network spinale indotti da stress ossidativo in wild type (WT) e cambiamenti nell’attività sinaptica dei circuiti premotori sottoposti ad uno stress infiammatorio in WT.
Il fine principale del mio lavoro era quello di capire il dialogo tra una condizione di stress generale ad il network spinale premotorio. A questo scopo, ho unito tecniche elettrofisiologiche e analisi di immunofluorescenza per caratterizzare gli interneuroni ventrali localizzati nel microcircuito spinale. Per raggiungere questo obiettivo ho sfruttato le colture organotipiche derivate dal midollo spinale di embrioni di topo che sono generalmente accettate come un buon modello per studiare l’attività premotoria neuronale e garantiscono un facile accesso sperimentale agli interneuroni (Avossa et al., 2003).
Nella prima parte della mia ricerca ho confrontato colture WT con colture transgeniche SOD1G93A, uno dei modelli di SLA maggiormente studiati. Nei network spinali in coltura, come già descritto in preparazioni acute ottenute a diversi stadi di sviluppo, c’è una progressiva velocizzazione delle correnti glicinergiche, rappresentata dalla riduzione del decay time constant (valore di tau) delle correnti sinaptiche durante la crescita delle fettine. Le colture WT e SOD1G93A presentano un diverso profilo di maturazione dato che nelle colture transgeniche questo processo di sviluppo è significativamente più marcato non solo nelle correnti postsinaptiche (PSCs) ma anche negli eventi in miniatura (mPSCs). Questa differenza nelle proprietà cinetiche delle correnti glicinergiche può essere fortemente ridotta dalla presenza di TBOA che diminuisce la sintesi del GABA. Questi risultati supportano l’ipotesi che nelle colture SOD1G93A ci sia un aumento del co-rilascio GABA/glicina portando alla conclusione che il rilascio sinaptico sia condizionato dalla presenza della mutazione ad uno stadio precoce dello sviluppo, prima di qualsiasi degenerazione neuronale evidente. Inoltre, ho supportato questo dato anche con risultati preliminari riguardanti la marcatura di GlyT2 e GAD65 (due marker per la glicina ed il GABA presinaptici rispettivamente). Infatti, le fettine spinali organotipiche SOD1G93A sembrano caratterizzate da un aumento delle sinapsi miste.
Successivamente, ho testato altri processi di stress del tessuto che potrebbero interferire con l’attività sinaptica e, conseguentemente, alterare l’attività del network. Dato che le fettine spinali sono preparazioni a lungo termine, ho testato incubazioni croniche con molecole chiave nei processi di stress: perossido di idrogeno (H2O2) per creare uno stress ossidativo e lipopolisaccaride (LPS) o una miscela di citochine (CKs: TNF-α, IL-1β and GM-CSF) per mimare uno stato infiammatorio. Per questo set di esperimenti ho usato un altro ceppo di topi privo di manipolazione genetica. Tutti questi trattamenti cronici aumentano la frequenza delle correnti mediate dai recettori AMPA; inoltre, uno stato infiammatorio è in grado di incrementare l’attività globale del network; il trattamento con LPS aumenta anche l’ampiezza delle correnti sinaptiche AMPA-mediate, sia spontanee che in miniatura, mentre le CKs accelerano il ritmo dei burst indotto dall’eliminazione farmacologica dell’inibizione sinaptica che accende il centro ritmogenico presente nel network spinale. Riassumendo, ho dimostrato che i trattamenti con questi fattori ambientali alterano la componente sinaptica, in questo caso eccitatoria, del network premotorio.
Nel complesso il mio lavoro ha evidenziato che una predisposizione genetica (nel caso della SLA familiare) e fattori ambientali di varia natura (ossidativi, infiammatori o di alterata SOD1) inducono cambiamenti nella trasmissione sinaptica e possiamo speculare sul fatto che queste alterazioni nel circuito premotorio possono cooperare in sinergia causando lo sviluppo di network inefficienti che concorrono a determinare la neurodegenerazione motoneuronale.<br>XXVI Ciclo<br>1985
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Patel, Leena Suman. "Functional changes in pyramidal neurons surviving an excitotoxic challenge in mouse organotypic slice cultures /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/10658.
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Isaacson, Erin. "Understanding the molecular basis of HPV-16 neoplastic progression using an organotypic raft model." Thesis, Open University, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522239.
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Meng, Weina. "Evaluation of a nanoparticle drug delivery vehicle in medulloblastoma and organotypic brain cell cultures." Thesis, University of Nottingham, 2006. http://eprints.nottingham.ac.uk/13933/.
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It has been widely reported that cell culture dimension and microenvironment influence cell proliferation, differentiation, and gene expression, which lead to different interactions between drug delivery systems and cells. The development in evaluation of drug delivery systems has reached the stage where investigations are now concentrating on intracellular uptake and subcellular localization of drug delivery systems.This thesis investigates the use of three-dimensional (3-D) tissue culture models to study how nanoparticles (NPs) may behave in vivo. Poly (glycerol-adipate) (PGA) NPs can degrade into glycerol and adipate, which are not having toxic and anyundesirable local or systemic effects in the host. Following on the initial physicochemical characterization of PGA NPs loaded with drug and fluorescent dyes, investigations moved on to the biological studies of NPs in various cell culture model, e.g. monolayer culture, 3-D culture models, and brain tumour invasion model. Particle size, surface charge, and hydrophobicity are important features affecting the amount of particles taken up by cells and intracellular localisation of particles. Thus, the physicochemical properties of drug and fluorescent dye loaded PGA NPs were assessed by Photon Correlation Spectroscopy, Laser Doppler Anemometry, and drug/fluorescent dye loading studies. These studies indicated that physicochemical properties of drug, fluorescent dyes and PGA polymer could influence drug /fluorescent dye loading, which results in different particle size and surface charge of PGA NPs. Quantitative and qualitative investigations into the influence of cell culture dimension on uptake of NPs by cells, both by confocal fluorescence microscopy and flow cytometry, revealed that DAOY cells took up NPs more effectively when in 3-D spherical aggregate culture than in 2-D monolayer culture while uptake of NPs by normal brain cells was lower in 3-D cell culture than that seen in 2-D monolayer culture. This resulted in intracellular fluorescence intensity about 6 times higher in DAOY aggregates than normal brain cell aggregates while in monolayer culture mixed brain cells took up 2 times as many NP as the DAOY cells. The results from studies of NPs migrating through aggregates and tissue slices also indicated that penetration ofNPs in 3-D culture models was affected by the structure of the interstitial compartment and composition of extracellular matrix. Microscopic investigation of the histology of a co-culture invasion model of DAOY aggregates and a organotypic brain slice confirmed that DAOY cells massively invaded into cerebellum slices after a 4-day co-culture while the invasion of DAOY cells were limited within cerebral cortex slices even after a 6-day co-culture. Selective uptake of NPs by host cells and brain tumour cells were also assessed in this 3-D brain tumour invasion model. It showed that most NPs were taken up by DAOY cells instead of brain cells.
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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.<br>Ph. D.
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Croft, Cara Louise. "Investigating the mechanisms underlying Alzheimer's disease using a novel organotypic brain slice culture model." Thesis, King's College London (University of London), 2016. http://kclpure.kcl.ac.uk/portal/en/theses/investigating-the-mechanisms-underlying-alzheimers-disease-using-a-novel-organotypic-brain-slice-culture-model(321a7046-c8e8-4c42-9abf-01792be7870c).html.
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Alzheimer's disease is a devastating progressive neurodegenerative disorder characterised by deposits of amyloid-β in extracellular plaques, intracellular neurofibrillary tangles comprising highly phosphorylated and aggregated tau species, synaptic dysfunction and neuronal death. Although several transgenic mouse models of Alzheimer's disease have been developed, in vivo studies using transgenic mice are time- and cost- consuming, and it is imperative that more ethically sustainable alternatives, which allow faster translation to the clinic, are developed. This thesis aims to determine if organotypic brain slice cultures from 3xTg-AD mice recapitulate key features of in vivo brain and can be used in mechanistic and pre-clinical Alzheimer's disease studies. Organotypic brain slice cultures prepared from 3xTg-AD mouse pups and maintained in culture for several weeks developed highly phosphorylated and high molecular weight tau species, increased amounts of β-amyloid and showed increased activation of cyclin-dependent kinase 5 over time. These biochemical changes closely recapitulate the molecular changes observed in in vivo models and post-mortem Alzheimer's disease brain. In addition, brain slices from wild-type and 3xTg-AD mice showed altered tau release characteristics, indicating a distinction in the mechanisms underlying physiological and pathological tau release. These data support the notion that brain slice cultures can be used to understand the mechanisms and pathways underlying the development and progression of Alzheimer's disease.
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Studer, Lorenz. "NGF increases neuritic complexity of cholinergic interneurons in organotypic cultures of neonatal rat striatum /." [S.l.] : [s.n.], 1994. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Cocchi, M. A. "THE MELATONIN PROTECTIVE ROLE IN AN ORGANOTYPIC MODEL OF SPINAL CORD INJURY SECONDARY DAMAGE." Doctoral thesis, Università degli Studi di Milano, 2016. http://hdl.handle.net/2434/351674.
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Spinal cord injury (SCI) is characterized to be a two-step process composed by the primary lesion consisting of the initial trauma and the secondary damage, characterized by multiple processes including inflammation, oxidative stress and cell death that lead to a significant expansion of the original damage and to an increase of the functional deficit. Among the aforementioned processes, the oxidative stress plays a significant role in pathophysiology of SCI. In this study, we evaluated the role of melatonin, potent antioxidant and immunomodulator indoleamin, on the oxidative stress, the tissue viability and the neuritic plasticity deriving from the gray matter in an experimental model of organotypic cultures. These cultures consisted of Sprague Dawley rat spinal cord slice treated with hydrogen peroxide (H2O2). In five experimental groups, A) Control Group (CTR) – Organotypic spinal cord slice culture (350μm); B) Stressed Group (H2O2) – Organotypic spinal cord slice culture (350μm) exposed to H2O2 (50 μM); C) Control Group treated with melatonin (10-5M) of 24 hours (CTR+MEL) – Organotypic spinal cord slice culture (350μm) treated with melatonin for 24 hours; D) Treated Group (H2O2+MEL-POST) – Organotypic spinal cord slice culture (350μm) exposed to H2O2 (50 μM) and treated after 24 hours with melatonin (10-5M) for 24 hours; E) Treated Group (H2O2+MEL-PRE) – Organotypic spinal cord slice culture (350μm) pre-treated with melatonin (10-5M) for 24 hours (50 μM) and exposed to H2O2 for other 24 hours.
We investigated the slice cellular death by propidium iodide (PI) assay, the slice vitality by MTT assay, the superoxide dismutase (SOD) and total thiols (SH) levels for the contrast to the oxidative stress, the neuronal (NeuN) and the synaptophysin (Syp) immunopositivity. Melatonin significantly decreased the number of dead cells, increased slice vitality, mainly in slices treated before H2O2 exposition. Melatonin enhanced SOD immunopositivity, contrasted total thiols decrease, attenuated Syp reduction and increased NeuN immunopositivity. Overall, these findings suggest that melatonin may exert a potentially beneficial effect upon the progression of SCI secondary damage, protecting the tissue from a further degeneration.
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Ireland, Kirsty Anne. "Development of whole brain organotypic slice culture to investigate in vitro seeding of amyloid plaques." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28707.
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A feature of prion disease and other protein misfolding neurodegenerative disease is the formation of amyloid plaques. Amyloid is commonly found in the brain of individuals who have died from prion disease and Alzheimer’s disease. The formation and purpose of amyloid in such diseases is poorly understood and it is not currently known whether the material is neurotoxic, neuroprotective or an artefact. Several methods are used to investigate the formation of amyloid both in vitro and in vivo. A cell free protein conversion assay has been optimised to gain insight into the protein misfolding pathway and prion infection has been introduced to a newly characterised whole brain organotypic slice culture model. Fibrillar, but not oligomeric, recombinant PrP species induce a seeding effect on amyloid formation in the protein conversion assay. Brain homogenate containing amyloid from a β-amyloid aggregation mouse model is demonstrated to have a similar effect to recombinant fibril seeds with a PrP substrate indicating a cross-seeding effect. A whole brain organotypic slice culture (BOSC) model has been developed and slices maintained in culture for up to 8 months. During this time slices remain viable with low levels of stress and thin down from 400μm to 30-50μm with morphological consequences. A prominent glial scar forms on the surface of the slice as a result of astrocyte activation and proliferation. The neuronal population decreases while the microglia have a consistent presence throughout time in culture. Replication of misfolded prion protein has been successfully demonstrated within whole BOSC following prion infection after 2 months in culture. The BOSC model represents an accessible short term in vitro model of the brain which can offer insights into protein misfolding in a complex multicellular context. Amyloid formation has been investigated in vivo using a β-amyloid misfolding mouse model following seeding with a range of recombinant protein and brain homogenate seeds. No seeding effect was observed in animals which had received intracerebral inoculations compared to control animals within the time frame of the experiment. A lack of overall amyloid within all animals at the final time point investigated suggests later time points are required for observation of seeding. The functional role of amyloid in protein misfolding neurodegenerative diseases remains unclear. From the cell free protein conversion assay oligomers do not form on the direct pathway towards amyloid in prion misfolding. BOSC provide an accessible and useful short term in vitro model which retains multiple characteristics of the brain. BOSC support replication of misfolded protein and amyloid formation therefore this model can now be utilised to investigate plaque growth and the effect of amyloid formation on surrounding cells. Results from these assays provide important information to guide future in vivo studies and aid the search for therapeutic intervention in these devastating diseases.
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Bonilla-Pons, Sergi A. "Setting up stem cell therapy for human retina: from organotypic cultures to cell fusion understanding." Doctoral thesis, Universitat de Barcelona, 2021. http://hdl.handle.net/10803/673448.
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Visual impairments and different retinopathies have been silently increasing in the modern society and they become a medical hurdle in need to be addressed. Müller glial cells (MGCs), in lower vertebrates, show regenerative potential for the retina, however in the mammals this capacity is lost. However, it has been demonstrated in mice that fusion between MGCs and adult stem cells result in the formation of hybrids which detain some potential to regenerate the lost retinal neurons.
In the present work we hypothesize that also into the human retina cell fusion between adult stem cells and retinae resident MGCs can enhance a process resulting into retina rescue and regeneration. In this thesis we first demonstrated that cell fusion can occur between human MGCs (hMGCs) and adult stem cells in the human retinal organotypic cultures. We then isolated the hMGCs and we fused them with either hematopoietic stem cells or with human mesenchymal stem cells adipose-derived (hMSC AD) in culture. We found that the activation of the Wnt/b-catenin pathway can induce reprogramming of the hybrids, which, in turn, can then undergo differentiation and acquire some electrophysiological response. Finally, we set up a microinjection system to transplant the hybrids into human retinal organoids to finally study the hybrid differentiation in vivo.
With the strategy here exposed, we believe that it might represent the first step towards a potential regenerative therapy for human retinae via cell fusion and reprogramming. These observations might be the basis to develop an innovative approach to address in the long run the unmet medical need of the visual impairments.
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Singh, Sunil. "Engineered Organotypic Breast Tumor Model for Mechanistic Studies of Tumor-Stromal Interactions and Drug Discovery." University of Akron / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=akron1616940345310981.
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CAMBINI, AIMONE. "Effetti della biostimolazione laser sul tessuto epiteliale per il differenziamento dello strato cheratinizzato: studio in vitro." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2013. http://hdl.handle.net/10281/43489.
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Background: The overall view of gingival augmentation techniques proposed in the international literature does not exclude a surgical component. The planning of one or more surgical phases with the consequent post-surgical discomfort and results not always predictable are required when setting up periodontal flaps combined eventually with free gingival grafts. The introduction in the last years of laser biostimulation has led to a less invasive approach, particularly in the treatment of periodontally compromised patients, limiting the surgical phase to seriously compromised cases, with regeneration techniques for the restoration of a correct periodontal tissue’s anatomy. Aim of this in vitro study is to establish the validity of laser biostimulation in order to develop the epithelial tissue’s keratinized layer, by stimulating fibroblasts-keratinocytes organotypic cultures obtained and fibroblasts and keratinocytes mono-cultures.
Materials and Methods: We created two groups (test and control) each one of them composed by 3 fibroblast cultures, 3 keratinocyte cultures and 3 organotypic cultures. We performed laser irradiation of test group at 50 J/cm2 of fluency with one application every 48 hours for a total of five applications. 48 hours from last laser application we investigated the presence and amount of keratins 5 and 8 by performing citofluorymetric and western blotting analyses.
Results: the analyses results showed an increase in keratin synthesis of the test groups’ cultures, particularly showing a remarkable increase in production of keratin 8 by test co-cultures.
Conclusions: laser biostimulation proved to considerably enhance keratin synthesis when applied with high energy doses and repeated applications to keratinocytes-fibroblasts co-cultures.
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Vautrin, Sandrine. "Investigating oligodendrocyte development and stability using organotypic hippocampal slices, confocal imaging and viral gene delivery methods." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111543.
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Myelin plays an essential function in the behaviour of higher organisms by increasing the speed of axonal conduction. Indeed, myelin deficiency or damage can lead to serious motor and sensory dysfunction. In the central nervous system, myelin is produced by a specialized macroglial cell known as the oligodendrocyte. Although much is known about oligodendrocytes with respect to their role in myelin production, many details regarding their development and maintenance still remain unclear. These details may be of great importance for fully understanding the fundamental properties of oligodendrocytes and for devising strategies for treating myelin-related diseases. In this thesis, we report the development of a system using organotypic hippocampal slices, viral gene delivery methods, immunostaining techniques and confocal imaging that can be used to study the properties of oligodendrocytes and myelin. This system preserves many features of the intact brain and can be used to investigate cells of the oligodendrocyte lineage at different developmental stages. Individual oligodendrocytes were targeted using this system and we showed that they can be induced to express various proteins, such as proteolipid protein and neurofascin-155, that localized to specific compartments of oligodendrocytes. This system was then used to address the importance of glutamate receptor signalling on myelin. Studies were performed at the light microscopic level using agonists and antagonists of ionotropic glutamate receptors. Myelin showed progressive pathology over the course of hours following exposure to glutamate receptor agonists and, interestingly, glutamate signalling was not essential for myelin maintenance over a 48 hour period. This thesis work thus describes the use of a novel system that can help analyze both the cellular and molecular aspects of oligodendrocyte development and maintenance.
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Smith, Donna Louise. "An organotypic slice culture assay to assess huntingtin aggregation in Huntington's disease R6/2 transgenic mice." Thesis, King's College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.406704.
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Athar, Saira. "Immortalization of human oral keratinocytes with defined genetic elements in the development of organotypic oral culture." Thesis, Queen Mary, University of London, 2013. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8690.
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Primary cell culture is limited by the increase in cellular levels of p16INK4a in response to an in vitro culture environment and, in conjunction with telomere shortening following cell division, presents a barrier to cellular proliferation. The use of transformed cell lines is limited for studies wherein the aim is to generate data akin to an in vivo environment as commonly such cell lines achieve their immortal benefits by down regulating important tumour suppressive mechanisms and inhibiting cell cycle checkpoints. Normal Human Oral Keratinocyte (NHOK) cells expressing shp16+hTERT were generated and compared to NHOK cells expressing Bmi1 +hTERT using an optimized retroviral transduction protocol and compared simultaneously to an epidermal control. Population doubling assessment of cell lines revealed that shp16+hTERT was not sufficient to extend replicative lifespan in the absence of p53 whilst cell lifespan extension was observed not only in cells expressing Bmi1+hTERT, but also in cells transduced with Bmi1 alone. Upon characterization, cells showed expression of p53 and responsiveness to UVB-induced apoptosis as demonstrated by an increase in p53 expression. NHOKBmi1+hTERT displayed adaptability to serum free culture when weaned into keratinocyte serum free media (KSFM) and retained the ability to stratify into multiple layers when supported by feeders on polycarbonate membrane inserts. The cell line NHOKBmi1+hTERT will be beneficial for in vitro studies looking to utilise an alternative to transformed or spontaneously derived cell lines and holds potential for further development and optimization into a well characterized SSE in a user friendly, and reproducible system for the testing for oral products.
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Dallacasagrande, Valentina. "Novel approaches to study the biomechanics of intact central nervous tissue." Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-163943.
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In nature, cells are not randomly clustered to form tissues. The tissue is a more complicated system with functions that go beyond what any single cell type could accomplish. While studying single-cell mechanics and dynamics is relevant from an investigative point of view, this approach loses, or fail to gather information about the tissue. The tissue investigated in this study is the neurosensory retina which seeing as extension of the brain is a very convenient model for the central nervous system due to its accessibility.
The retina is constantly subjected to different mechanical stresses from development to adulthood. Although the majority of the phenomena where mechanical stresses are involved are well-studied, the mechanics behind them is not well understood. However, knowledge about the ability of the retina to adjust to mechanical stresses is essential, for example, for improving retinal surgery.
Establishing a method to mechanically probe the retina is a challenge due to the extremely delicate nature of this multilayered neural tissue and to the short-time survival ex vivo. The organotypic tissue culture is a powerful tool because it allows to maintain with high accuracy the complex multicellular anatomy and the microenvironment of the original tissue. One of the limitations of the organotypic culture techniques has been until recently due to the ability to use only post-natal/juvenile tissues for long-term culture. The importance of using adult tissue is incontestable when the investigation focuses on age-related pathologies such as vitreous shrinkage or macula degeneration.
In this work, TiO2 nanotube arrays are presented as the innovative substrate for long-term organotypic culture of adult neural tissue. The retinal whole-mount of adult guinea pig and the brain slices of adult mouse were cultures for 14 days without showing any sign of edema or swelling. Furthermore, in order to study the behavior of the retinal tissue under shear stress new set-ups were designed. For the first time, the behavior of the retinal layers were observed showing that the retina does not act as an homogeneous material in response to an applied stress. The methods developed here can be used for future quantitative studies, to provide an exact knowledge of retinal biomechanics which will help retinal surgeons to optimize their methods.
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McBain, Christopher J. "An electrophysiological investigation of the spontaneously arising epileptiform activity seen in organotypic cultures of neonate rat hippocampus." Thesis, University of Cambridge, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.293815.
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Stubblefield, Park Samantha Renee. "Organotypic brain explants reveal an interleukin-12 / interferon-γ / T-cell dependent clearance of measles virus infection". Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1297280439.
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Sullivan, Aideen Margaret. "The use of organotypic cultures of rat cerebellum for the study of neuromodulatory interactions in the mammalian brain." Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243197.
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Jagger, Elizabeth. "The effects of kainic acid in hippocampal organotypic cultures : a chronic in vitro model of temporal lobe epilepsy." Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269697.
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Barnhart, Kirstin Faye. "In vitro and in vivo analysis of differential gene expression between normal norfolk terrier dogs and those with an autosomal recessive mutation in KRT10." Texas A&M University, 2004. http://hdl.handle.net/1969.1/2671.
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Natural diseases caused by keratin mutations are rare and have only been reported in humans. We have recently identified a heritable skin disorder in Norfolk terriers caused by a mutation in KRT10. Affected dogs have a tendency to form shallow erosions or blisters following mild trauma, which is first noted after the birthing process. As the dogs age, they display generalized hyperpigmentation and scaling that is most severe in the axillary and inguinal regions. The main histologic and ultrastructural features include: marked hyperkeratosis, epidermal hyperplasia, prominent vacuolation of the upper suprabasal layers, eosinophilic intracytoplasmic aggregates (keratin bundles), numerous and frequently enlarged keratohyaline granules, and epidermal hyperplasia. Analysis of an extended pedigree through seven generations confirmed an autosomal recessive mode of inheritance. The keratin 10 mutation was defined as a G-T point mutation in intron 5 that affected splicing at the boundary of exon 4 and intron 5. The primary outcome of the mutation was a 35 bp deletion in exon 4 caused by use of a cryptic splice site. Real-time PCR quantitation of KRT10 confirmed that this mutation led to premature mRNA decay and an average 35-fold decrease in KRT10 message.
Organotypic cell culture techniques were used to establish in vitro models for normal and affected Norfolk terriers. After 21 days of culture, normal epidermis was cornified with a compact and multifocally parakeratotic stratum corneum. Affected epidermis largely reproduced the expected morphologic alterations. Immunoblotting and immunohistochemistry for keratin 10 protein and real-time PCR quantitation of KRT10 message showed significantly less keratin expression in vitro than in vivo suggesting that the differentiation program in vitro underwent significant alterations.
A diagnostic PCR assay was established for detection of the carrier state. Global analysis of gene expression between normal, carrier and affected dogs was performed with DermArray cDNA microarrays. Affected and carrier dogs showed differential regulation of 320 and 298 genes, respectively, between normal dogs. In affected dogs, 217 were upregulated and 103 were downregulated. In carrier dogs, 222 were upregulated and 76 were downregulated. 72 genes (65 upregulated, 7 downregulated) were altered in both affected and heterozygous dogs.
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Wills, Lauren Raquel. "Investigating Induced Pluripotent Stem Cells for Tissue Engineering and Hepatotoxicity Applications." Thesis, Virginia Tech, 2019. http://hdl.handle.net/10919/101006.
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Induced pluripotent stem cells (iPSCs) can be differentiated into multiple cell types in the body while maintaining proliferative capabilities. The generation of human iPSC-derived hepatocytes (iPSC-Heps) has resulted in a new source for hepatic cells. The current available options for human hepatocytes are primary human hepatocytes (PHHs) and cell lines. PHHs isolated from healthy human donors are difficult to obtain, while cell lines exhibit reduced hepatotoxic sensitivity. iPSC-Heps are being investigated as an alternative option as they are derived from a continuous, stable source and are able to maintain their original donor genotype, which opens the door for patient-specific studies. iPSC-Heps show promise for utilization in tissue engineering, hepatotoxicity studies as well as screening for patient-specific therapeutics. Various reports have concluded that iPSC-Heps exhibit reduced hepatocyte function in comparison to PHHs. Prior reports on iPSC-Heps have focused on improving their adult phenotype functions through variations in differentiation protocols or by altering their in vitro culturing environment. This thesis focuses on incorporating hepatic non-parenchymal cells to more closely mimic the tissue and cell architecture found in the liver tissue. We designed and assembled a 3D iPSC-Hep model that integrates liver sinusoidal endothelial cells, with the goal of achieving functional maturity. Hepatotoxicants were administered to our models and various hepatic markers were measured to analyze the toxic response. This work demonstrates the need for the inclusion of hepatic non-parenchymal cells in iPSC-derived liver tissues, specifically for hepatotoxicity applications.<br>Master of Science
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Werner, Renata Verfasser], та Mathias [Akademischer Betreuer] [Jucker. "Modeling the prion aspect of cerebral β-amyloidosis in organotypic slice cultures and mice / Renata Werner ; Betreuer: Mathias Jucker". Tübingen : Universitätsbibliothek Tübingen, 2018. http://d-nb.info/1168728371/34.
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Jevtić, Marijana [Verfasser]. "Fine-Tuning of Organotypic Skin Equivalents for Preclinical Research and Their Utilization to Study Epidermal-Dermal Crosstalk / Marijana Jevtić." Berlin : Freie Universität Berlin, 2021. http://d-nb.info/1229436650/34.
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Nash, Claire. "Development of an organotypic 3D in vitro model of normal human breast tissue : a tool for cancer initiation studies." Thesis, University of Leeds, 2014. http://etheses.whiterose.ac.uk/6845/.
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The mechanisms involved in breast cancer initiation are not well understood. This may in part be due to a lack of an in vitro model that faithfully recapitulates the morphology, phenotype and in vivo architecture of the normal human mammary gland. Most in vitro models of normal breast have relied on the use of reconstituted basement membrane gels to induce luminal epithelial cell polarity and have neglected the role of myoepithelial cells and fibroblasts in this process. The aim of this thesis was to develop a three dimensional in vitro culture system of normal breast which included three of the major functional cell types of breast embedded in a more physiologically relevant collagen I matrix. It was then sought to use the system to investigate the mechanisms behind breast cancer initiation via genetic manipulation of well-known oncogenes and tumour suppressors involved in breast cancer progression. To achieve this, myoepithelial cells (Myo1089, originally isolated from breast reduction mammoplasty sample, gift of Dr Mike O’Hare) and fibroblasts (isolated and immortalised from breast reduction mammoplasty samples collected with ethical approval in house) were characterised by immunofluorescence to assess their suitability. Following characterisation, these were virally transfected with Turbo Green Fluorescent Protein (tGFP) and dsRed protein respectively to enable tracking. Three-dimensional tri-cultures were established in collagen I and included the non-tumorigenic luminal epithelial cell line HB2 with GFP Myo1089 cells and dsRed fibroblasts. Cells were cultured for three weeks in Transwell™ cell culture inserts. Following fixation these were analysed by haematoxylin and eosin staining, confocal microscopy and immunohistochemistry. Morphology and immunostaining profiles were compared to sections of a normal human in vivo breast tissue specimen. Immunohistochemical characterisation using the following antibodies: E-cadherin, epithelial membrane antigen, vimentin, laminin 5, collagen IV plus luminal and basal cytokeratins, demonstrated polarised epithelial structures with lumen formation and basement membrane production with a similar immunostaining profile to normal breast tissue. The importance of including myoepithelial cells and fibroblasts in maintaining these structures was demonstrated. We established this model was amenable to genetic engineering by overexpressing HER2 and HER3 in HB2 cells, and knocking out ERβ1 in Myo1089 cells and DOCK4 in fibroblast cell lines using siRNA/shRNA techniques respectively. These were included in separate models with morphological and phenotypic effects determined by haematoxylin and eosin staining, immunohistochemistry and quantification of HB2 structures formed. We further investigated the intracellular signalling cascades stimulated by heregulin in order to validate our findings upon overexpression of HER2 and HER3 and to investigate the cancer initiation potential of heregulin in the breast. In summary, an in vitro model of normal breast tissue that includes three of the major functional breast cell types cultured in a physiologically relevant three dimensional matrix has been developed. The morphology and protein expression profile of the model was validated against a human breast tissue specimen and confirmed that it is a suitable model of normal breast. The model proved to be reproducible, suitable for experimentation using genetic engineering and cell behaviour could be easily visualised using standard laboratory techniques. To conclude, this is a robust in vitro model of normal breast tissue offering an alternative cost-effective method of studying genes and processes involved in breast cancer initiation.
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Müller, Mareike [Verfasser], Horst-Werner [Akademischer Betreuer] Korf, Horst [Akademischer Betreuer] Stöcker, and Marco [Akademischer Betreuer] Durante. "Effects of ionizing radiation on organotypic slice cultures / Mareike Müller. Gutachter: Horst Stöcker ; Marco Durante. Betreuer: Horst-Werner Korf." Frankfurt am Main : Univ.-Bibliothek Frankfurt am Main, 2013. http://d-nb.info/104397816X/34.
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Olivieri, Dario. "New Spinal cord models: Characterization of Excitotoxicity and Neuroprotection." Doctoral thesis, SISSA, 2015. http://hdl.handle.net/20.500.11767/3892.
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Group III metabotropic glutamate receptors (mGluR III) are known to decrease glutamate release and to play an important role in controlling pain as documented in neuropathic pain models. Much less is known about their potential neuroprotective effect against excitotoxicity that is considered important for damage onset of spinal cord injury. Using rat spinal cord organotypic slices model, we investigated if mGluR III receptor activation might contrast excitotoxic cell death evoked by kainic acid (0.1 mM) applied for 1h and followed by wash for further 24h. The specific agonist of mGluR III receptors L-(+)-2-amino-4-phosphonobutyric acid (L-AP4; 1 µM) was either co-applied with kainic acid or administered during washout. Cell death was quantified in terms of percentage of pyknotic nuclei, total number of neurons, motoneurons and astrocytes.
Furthermore we developed for future long-term studies an in vitro model of Spinal Cord Isolated from newborn rats maintained for 3 days in medium. We characterize this model using both immunohistochemistry and electrophysiological recordings.
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Sygnecka, Katja. "Organotypic brain slice co-cultures of the dopaminergic system - A model for the identification of neuroregenerative substances and cell populations." Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-188897.
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The development of new therapeutical approaches, devised to foster the regeneration of neuronal circuits after injury and/or in neurodegenerative diseases, is of great importance. The impairment of dopaminergic projections is especially severe, because these projections are involved in crucial brain functions such as motor control, reward and cognition. In the work presented here, organotypic brain slice co-cultures of (a) the mesostriatal and (b) the mesocortical dopaminergic projection systems consisting of tissue sections of the ventral tegmental area/substantia nigra (VTA/SN), in combination with the target regions of (a) the striatum (STR) or (b) the prefrontal cortex (PFC), respectively, were used to evaluate different approaches to stimulate neurite outgrowth: (i) inhibition of cAMP/cGMP turnover with 3’,5’ cyclic nucleotide phosphodiesterase inhibitors (PDE-Is), (ii) blockade of calcium currents with nimodipine, and (iii) the co-cultivation with bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs). The neurite growth-promoting properties of the tested substances and cell populations were analyzed by neurite density quantification in the border region between the two brain slices, using biocytin tracing or tyrosine hydroxylase labeling and automated image processing procedures. In addition, toxicological tests and gene expression analyses were conducted.
(i) PDE-Is were applied to VTA/SN+STR rat co-cultures. The quantification of neurite density after both biocytin tracing and tyrosine hydroxylase labeling revealed a growth promoting effect of the PDE2A-Is BAY60-7550 and ND7001. The application of the PDE10-I MP-10 did not alter neurite density in comparison to the vehicle control.
(ii) The effects of nimodipine were evaluated in VTA/SN+PFC rat co-cultures. A neurite growth-promoting effect of 0.1 µM and 1 µM nimodipine was demonstrated in a projection system of the CNS. In contrast, the application of 10 µM nimodipine did not alter neurite density, compared to the vehicle control, but induced the activation of the apoptosis marker caspase 3. The expression levels of the investigated genes, including Ca2+ binding proteins (Pvalb, S100b), immediate early genes (Arc, Egr1, Egr2, Egr4, Fos and JunB), glial fibrillary acidic protein, and myelin components (Mal, Mog, Plp1) were not significantly changed (with the exception of Egr4) by the treatment with 0.1 µM and 1 µM nimodipine.
(iii) Bulk BM-MSCs that were classically isolated by plastic adhesion were compared to the subpopulation Sca-1+Lin-CD45--derived MSCs (SL45-MSCs). The neurite growth-promoting properties of both MSC populations were quantified in VTA/SN+PFC mouse co-cultures. For this purpose, the MSCs were seeded on glass slides that were placed underneath the co-cultures. A significantly enhanced neurite density within the co-cultures was induced by both bulk BM-MSCs and SL45-MSCs. SL45-MSCs increased neurite density to a higher degree. The characterization of both MSC populations revealed that the frequency of fibroblast colony forming units (CFU-f ) is 105-fold higher in SL45-MSCs. SL45-MSCs were morphologically more homogeneous and expressed higher levels of nestin, BDNF and FGF2 compared to bulk BM-MSCs.
Thus, this work emphasizes the vast potential for molecular targeting with respect to the development of therapeutic strategies in the enhancement of neurite regrowth.
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Barth, Melanie [Verfasser]. "Organotypic slice culture models for induced alpha-synucleinopathy and exploration of the potential role of microglia in pathogenesis / Melanie Barth." Tübingen : Universitätsbibliothek Tübingen, 2023. http://d-nb.info/1238595006/34.
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Aldarwesh, Amal. "Oxygen and glucose deprivation on human Müller cells (MIO-M1) and human organotypic retinal cultures (HORCs) in relation to glaucoma." Thesis, University of East Anglia, 2015. https://ueaeprints.uea.ac.uk/58555/.
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Purpose: The purpose of this research was to investigate the effect of glaucoma-related insults, specifically oxygen and/or glucose deprivation (OGD) on the survival and genes expression of human Müller cells (MIO-M1), and retinal ganglion cells (RGCs) using the human organotypic retinal culture (HORC) model. Methods: MIO-M1 cells and HORCs were exposed to different levels of OGD using a custom-built chamber to control oxygen levels. Cell survival was evaluated using MTS and LDH assays while RGC death in HORCs was investigated using NeuN immunohistochemistry and TUNEL-labelling. Expression of genes of interest was assessed using QRT-PCR. Results: Reduced levels of oxygen and glucose (1.11mMglucose/4%O2) caused proliferation of MIO-M1 cells. Full deprivation of glucose caused cell death, but full hypoxia did not affect survival. In HORCs, glucose deprivation and OGD, but not oxygen deprivation alone, caused loss of RGCs. Different levels of OGD regulated expression of genes associated with angiogenesis, glial activation, excitotoxicity and neuroprotection in MIO-M1 cells and HORCs. VEGF expression significantly increased in MIO-M1 cells and HORCs treated with full OGD, and VEGF protein was secreted under reduced levels (1.11mMglucose/4%O2). Secretion of VEGF in MIO-M1 cells and HORCs was also increased under conditions of raised glucose. The PKCβ inhibitor LY333531 decreased VEGF secretion under conditions of raised glucose and hypoxia. Co-culture of MIO-M1 cells resulted in more damage/apoptosis to HORCs and reduced RGCs survival. Conclusions: Use MIO-M1 cells and the HORC model were effective in studying the effect of OGD in relation to glaucoma. Glucose rather than oxygen was the key survival factor for RGCs and MIO-M1 cells. The secreted factors by Müller cells could have protective and detrimental effects on RGC survival. Investigation of mechanisms using these models may be of benefit in development of potential therapeutic interventions for retinal neurodegenerative diseases including glaucoma.
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Linnemann, Jelena [Verfasser], and Magdalena [Akademischer Betreuer] Götz. "An organotypic assay for the quantification and characterization of regenerative primary human mammary epithelial cells / Jelena Linnemann ; Betreuer: Magdalena Götz." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1154683699/34.
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Niel, Olivier. "Développement vasculaire rénal in vivo et ex vivo : vers la bio-ingénierie rénale." Thesis, Nice, 2014. http://www.theses.fr/2014NICE4023.
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Chez la souris, la néphrogenèse débute par l'apparition du blastème metanéphrogène à 9.5 dpc. Une transition mésenchymo-épithéliale, comportant 5 étapes, débute a 11.5 dpc et aboutit au rein mature, composé de 3 structures : glomérules, tubules, et capillaires glomérulaires. Les étapes initiales du développement rénal peuvent être récapitulées en culture ex vivo; toutefois, l'organogenèse terminale et la maturation rénale sont incomplètes, et les structures rénales obtenues ex vivo ne sont pas fonctionnelles. Une étude du développement vasculaire in vivo au cours du développement rénal montre une angiogenèse (cellules Pecam-1 positives) et une vasculogenèse (cellules VEGFR-1 positives) précoces, dès 10.5 dpc. Une analyse quantitative par qRT-PCR confirme le rôle de Hif1α et VEGF dans la vasculogenèse rénale. En outre, la voie PGC1α, inductrice de VEGF indépendante de HIF, est activée en conditions hypoxiques. Pour améliorer le développement vasculaire rénal ex vivo, nous proposons un modèle de culture avec micro-perfusion rénale. L'étude morphologique par immunofluorescence des reins après culture micro-perfusée montre une survie tissulaire normale (TUNEL), et une intégrité anatomique (Néphrine, Cytokératine, WT1), en particulier vasculaire (Pecam-1). Une perfusion de vivo-morpholinos WT1 aboutit à une perte d'expression de WT1, confirmant le caractère fonctionnel de notre modèle. En conclusion, nous montrons le rôle précoce de l'angiogenèse et de la vasculogenèse au cours du développement rénal ; nous identifions le rôle de PGC1α dans la vasculogenèse rénale en conditions hypoxiques, et nous proposons une nouvelle technique de culture rénale ex vivo<br>In mice, nephrogenesis starts with the formation of the metanephric mesenchyme, at e9.5 dpc. A mesenchymal epithelial transition, consisting of 5 steps, starts at e11.5 dpc, and leads to a mature kidney, composed of 3 main structures: glomeruli, tubules, and capillaries. The initial steps of renal development can be recapitulated ex vivo; however, terminal organogenesis and maturation are impaired, and the explants are not functional. A study of vascular development in vivo during renal development shows that angiogenesis (Pecam-1 positive cells) and vasculogenesis (VEGF-R1 positive cells) occur early, at e10.5 dpc. A quantitative analysis, by qRT-PCR, shows that Hif1α and VEGF play a major role in renal vasculogenesis. Moreover, the PGC1α signaling pathway, a HIF independent VEGF inductor, is activated under hypoxic conditions. To improve ex vivo vascular development, we propose a novel culture technique, with micro-perfusion of the explant. A morphologic analysis of the kidneys obtained by micro-perfused cultures shows no apoptosis (TUNEL), a conserved parenchymal structure (Nephrin, Cytokeratin, WT1), and a proper vascular development (Pecam-1). A micro-perfusion of WT1 vivo-morpholinos leads to a decrease in WT1 expression, thus validating our model. In conclusion, we showed the early role of angiogenesis and vasculogenesis in renal development, we analyzed PGC1α role in hypoxic kidney cultures, and we proposed a novel kidney culture model
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Wang-Lauenstein, Lan [Verfasser]. "Assessment of chemical-induced local irritation and inflammation in organotypic lung tissue model : precision-cut lung slices (PCLS) / Lan Wang-Lauenstein." Hannover : Technische Informationsbibliothek (TIB), 2017. http://d-nb.info/1128673576/34.
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Verheyen, Leonie Katharina [Verfasser]. "Development of Organotypic Skin Disease Equivalents Mimicking Hallmarks of Atopic Dermatitis for Basic Research and Preclinical Drug Evaluation / Leonie Katharina Verheyen." Berlin : Freie Universität Berlin, 2018. http://d-nb.info/1176641182/34.
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Schmohl, Michael [Verfasser], and Stefan [Akademischer Betreuer] Stevanovic. "Analyzing biological activity of drugs- Inter- and intracellular signaling analysis within an organotypic co-culture system / Michael Schmohl ; Betreuer: Stefan Stevanovic." Tübingen : Universitätsbibliothek Tübingen, 2011. http://d-nb.info/1162699302/34.
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