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1
Mendes, Anabela Lindo dos Santos. "Cultura in vitro de embriões imaturos em ameixeira europeia." Master's thesis, Universidade de Évora, 1997. http://hdl.handle.net/10174/12550.
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Foram colhidos semanalmente, desde os 7 aos 70 dias após a polinização (DAP), frutos de Prunus domestica cv. ‘Rainha Cláudia Verde' provenientes de polinização livre. Com o objectivo de obter plântulas, foram extraídos os óvulos e/ou embriões e os eixos embrionários e testados três meios de cultura base, adicionando ou não diferentes tipos de reguladores de crescimento (auxina, giberelina e citocinina) e amino-ácidos (caseína hidrolisada 500 mg/l), tendo-se também variado a concentração de sacarose (2 e 10%). Foi ainda testada a técnica de perfuração da testa do óvulo e a influência do frio (30 dias a 4°C), na quebra de dormência dos embriões e eixos embrionários. Os resultados mostram que, aos 49 DAP quando comparámos a cultura do embrião dentro do óvulo perfurado, com a cultura do embrião isolado no meio de cuhura, verificámos que esta última conduziu a um melhor desenvolvimento do embrião e a percentagens de sobrevivência mais elevadas. Para os embriões no início da fase cotiledonar (49 DAP; PF1=11), o meio de cultura de Chée & Pool (1987) com 2% Sacarose +Caseína Hidrolisada (500 mg/1), demonstrou ser o mais adequado para o crescimento e sobrevivência dos embriões, quando comparado com os meios Nitsch & Nitsch (1969) e Stewart & Hsu (1977). Os embriões colocados neste meio de cultura apresentavam um comprimento médio inicial de 1,62 mm e atingiram em média os 5,31 mm, ao fim de 60 dias em cultura. No entanto, só foi possível obter plântulas a partir de embriões em plena fase cotiledonar (56 DAP; PF1=44). Neste estádio de desenvolvimento, o frio teve um efeito positivo tanto na percentagem de germinação como na qualidade das plântulas obtidas. Para os embriões em plena fase cotiledonar o meio de cultura Nitsch & Nitsch (1969) com 2% Sacarose +BAP (0,1mg/l.) +2,4-D (0,1mg/l), mostrou ser o mais adequado para a germinação, contudo, as plântulas obtidas apresentaram baixa qualidade.
### Abstract - The fruits of Prunus domestica cv. `Rainha Cláudia Verde' subject to opera pollination were picked weekly, from 7 to 70 days after pollination (DAP), with the aim of obtaining seedlings; the ovules and/or embryos and axis were extracted and placed in three different media with different concentrations of growth regulators (auxin, cytokinin and gibberellin), acid amino (casein hidrolysate 500 mg/l) and sacarose (2 and 10%). The ovule perforation technique and cold treatment (30 days at 4°C) were also applied to break down dormancy of the embryos and the embryonic axis. The results show that, compared to the ovule culture, the isolated embryo culture, at 49 DAP, gives a better embryo growth and a higher percentage of survival. The most adequate mediam for the growth and survival of the embryos at the early cotiledonar stage (49 DAP, PF1=11) proved to be the Chée & Pool medium (1987) with 2% sacarose+ Casein Hidrolysate (500 mg/l), as compared to the Nitsch & Nitsch medium (1969) and Stewart & Hsu medium (1977). The embryos that were placed in this medium initially had an average length of 1.62mm and after 60 days in the medium they had obtained an average length of 5.31mm. However, it was only possible to obtain seedlings from embryos at the cotiledonar stage (56 DAP, PF1=44). At this stage of development, the cold treatment had a positive effect, both for the percentage of germination and for the quality of the seedlings. For the embryos at the cotiledonar stage the Nitsch & Nitsch medium (1969) with 2% sacarose + BAP(0.1 mg/1)+ 2,4-D(0.1 mg/1) proved to be the most successful one for germination, however, the quality of these seedlings was poor.
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Langan, Laura. "Fish intestinal cultures for ecotoxicological studies : in vitro and primary culture models." Thesis, University of Plymouth, 2017. http://hdl.handle.net/10026.1/9486.
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Ecotoxicity testing of chemicals for environmental risk assessment is an area where a high number of vertebrates are used across a variety of industrial sectors. The application of the 3Rs in toxicity testing using fish address both the ethical and societal concerns around this issue in addition to the increasing legislative requests for the incorporation of animal alternatives. This thesis aims to highlight the potential of 3D cell culture models to "bridge the gap" between in vitro and in vivo screening procedures for testing of chemicals with the potential to persist or bioaccumulate thereby improving the predictive power of screening procedures. This thesis examines two alternative methods for their potential use as an intestinal based toxicokinetic tool for environmental risk assessment, utilising an in vitro fish cell line replacement tool (RTgutGC). In addition, for the first time a new intestinal primary cell culture based model was developed to address both intestine region specific response (pyloric, anterior, mid and posterior) and size related adaptability to toxins. Paramagnetic oximetry was used to measure oxygen content within 3D structures (spheroids) in order to better understand the microenvironment of these culture models. Using histology, immunohistochemistry, transepithelial electrical resistance (TEER), transmission electron microscopy (TEM), metabolic, fluorescence and gene expression assays, the comparability of this system to native intestinal response was established. Following exposure to carefully chosen environmental contaminants (Benzo[a]pyrene and Copper), the RTgutGC cell line demonstrated comparable responses to existing literature in terms of uptake, metabolism, DNA damage and the presence an equivalent saturable level. Primary enterocytes cultured on transwell inserts remained viable for upto six weeks, with permeability and metabolic activity comparable to native tissue (both in vitro and ex vivo). Taken in combination, these features of enterocytes represent a profile more closely representative of the intestine then the widely used "gut sac" method. With the potential advantages of incorporating complexity at differing levels (connective tissue layer, intestinal bacteria biome), the intestinal models described offer the potential to screen highly persistent toxins which may require prolonged incubation, in addition to the exploration of complex experimental designs which minimise animal usage (uptake, depuration, uptake). As a consequence, the models developed within this thesis significantly enrich the emerging fish based in vitro testing strategies.
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Czechowiak, Caty. "Culture in vitro de méristèmes de Pelargoniums." Lille 1, 1988. http://www.theses.fr/1988LIL10064.
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Contrairement à la production massive de boutures contribuant à augmenter l'extension des virus et bactéries (Xanthomonas), la culture de méristèmes de pélargonium ensemencés sur du milieu de base additionné de faibles quantités hormonales peuvent régénérer des plantes exemptes de maladies. Cependant en cas de trop fortes concentrations en hormones, le méristème peut produire un cal organogène générateur d'une dérive génétique. La méthode basée sur deux variétés: super rose (P. Hederaefolium) et topscore (P. Hortorum) a ensuite pu être généralisée.
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Nelson, T. J. "In vitro culture of Theobroma cacao L." Thesis, University of Nottingham, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.376391.
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Bishop, Rebecca Louise. "Pneumocystis carinii : approaches to in vitro culture." Thesis, London School of Hygiene and Tropical Medicine (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244726.
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Latif, Sjafrul. "In vitro culture of ginger and macadamia." Thesis, Queensland University of Technology, 2000.
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Najm, Nour Addeen. "In vitro culture studies of tick cell lines." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-146638.
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Tascan, Ayse. "In vitro liquid culture systems of Scutellaria species." Connect to this title online, 2007. http://etd.lib.clemson.edu/documents/1181251555/.
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Kaparakis, Georgios. "In vitro culture of pepper (Capsicum annuum L.)." Thesis, University of Nottingham, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297989.
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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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Saleil, Véronique. "Développement "in vitro" des apex isolés à partir de deux espèces d'igname, Dioscorea alata et Dioscorea trifida." Montpellier 2, 1986. http://www.theses.fr/1986MON20091.
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Shahabeddin, Lili. "Développement et caractérisation d'une "peau reconstruite" in vitro." Lyon 1, 1991. http://www.theses.fr/1991LYO1T026.
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Antonetti, Philippe. "Contribution à l'étude de la culture in vitro et de la vitro-variation du genre populus." Nancy 1, 1990. http://www.theses.fr/1990NAN10409.
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La première partie de ce travail consiste en une mise au point et une optimisation des techniques de culture in vitro du peuplier pour différents clones des sections Aigeiros, Tacamahaca et Leuce : obtention et culture de cals (pour 35 clones) ; néoformation de bourgeons sur les cals et régénération de la plante entière (pour 35 clones) ; micropropagation des clones originels et des néoformations dérivées de cals (pour 34 clones) ; obtention et culture de suspensions cellulaires (pour 6 clones) ; obtention et culture de protoplastes (pour 6 clones), la culture jusqu'au stade micro-cal reste à maitriser. La seconde partie concerne la mise en évidence de la vitro-variation. 1092 plants ont été régénérés à partir de cals de différents clones. 39 variants présentent des morphologies foliaires ou des ports différents. On ne note aucune variation à l'égard de la résistance à deux rouilles foliaires (Melampsora larici-populina et M. Allii-populina). Par contre, 5 plants issus d'une lignée de cals d'un clone de grisard devenue plus résistante au filtrat de culture d'hypoxylon mammatum (après culture des cals en présence de ce filtrat), présentent également une résistance accrue à ce filtrat. L'analyse en cytométrie en flux de nombreux variants montre que la plupart sont devenus tetraploides, quelques plants présentant des morphologies très modifiées sont restés diploides. L'analyse de quelques cals nous permet de signaler qu'ils sont généralement tétraploides
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Musselmann, Kurt. "Developing culture conditions to study keratocyte phenotypes in vitro." [Tampa, Fla] : University of South Florida, 2006. http://purl.fcla.edu/usf/dc/et/SFE0001726.
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Crisell, P. D. "HIV directed ribozymes in vitro and in cell culture." Thesis, University of Oxford, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306560.
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Esna-ashari, Mahmood. "In vitro culture and frost tolerance studies in Solanum." Thesis, University of Salford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360439.
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Rowlett, Karen. "In vitro and in ovo culture of fowl embryos." Thesis, University of Reading, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262850.
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Rosell, R. S. "Incubation of crustacean eggs in vitro." Thesis, University of Liverpool, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377136.
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Javouhey, Marc. "Application industrielle de la culture in vitro : cas de l'asperge (Asparagus officinalis L.)." Grenoble : ANRT, 1985. http://catalogue.bnf.fr/ark:/12148/cb37594828m.
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Batista, Mariana Raposo. "Desenvolvimento de um sistema de cultura in vitro de células lúteas bovinas para estudar as interacções com embriões em co-cultura." Master's thesis, Universidade Técnica de Lisboa. Faculdade de Medicina Veterinária, 2011. http://hdl.handle.net/10400.5/4325.
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Dissertação de Mestrado Integrado em Medicina VeterináriaO objectivo deste trabalho foi o desenvolvimento de um sistema de cultura in vitro de células lúteas bovinas, compatível com o sistema de cultura in vitro de embriões bovinos, para estudar as suas interacções, relevantes para o estabelecimento da gestação nos mamíferos. As células foram obtidas pela digestão de corpos lúteos (CL) pela colagenase, seguida de centrifugações em gradiente de Percoll® e centrifugações a velocidades decrescentes. Foram estudados os efeitos do estadio do CL do qual provêem as células lúteas, meio de cultura, concentração de soro no meio de cultura, tensão de oxigénio na atmosfera de cultura, criopreservação e cobertura da cultura com óleo mineral sobre a produção de progesterona (P4) pelas células lúteas em cultura. A P4 foi quantificada por radioimunoensaio. Verificou-se que o estadio do CL teve efeito significativo na P4 produzida, assim como a refrescagem do meio para as células provenientes do estadio early. Não se verificaram efeitos significativos dos restantes factores estudados. O óleo reduz cerca de 50× a P4 quantificada no meio. O sistema de cultura in vitro aqui desenvolvido apresenta as condições requeridas para a co-cultura de embriões.
ABSTRACT - Development of an in vitro culture system for bovine luteal cells which allow the study of embryo interactions in co-culture - The objective of this work was to develop an in vitro culture system for bovine luteal cells, compatible with embryo in vitro culture systems, to allow the study of their interactions, which are relevant for the establishment of mammalian pregnancy. The cells were obtained by collagenase digestion of the corpus luteum (CL), followed by Percoll® gradient centrifuges and decreasing speed centrifuges. The effects of the stage of the CL of origin, culture medium, serum concentration in the culture medium, oxygen tension in the culture atmosphere, cryopreservation and mineral oil overlaying of the culture wells were evaluated on the luteal cells’ ability to produce progesterone (P4). P4 was quantified by radioimmunoassay. From the above, the effects of the stage of the CL and of the refreshing of the culture medium in the cells of the early CL were significant. Oil overlaying reduced about 50× the P4 quantified in the medium. The luteal cell in vitro culture system here developed is able to support the required conditions for embryo co-culture sessions.
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Oliveira, Anna Cecília Bezerra de. "Derme reconstituída (equivalente) in vitro." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/82/82131/tde-03102017-155953/.
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Um dos desafios atuais da engenharia de tecidos é o desenvolvimento de biomateriais substitutos e/ou equivalentes que mimetizem o tecido normal. Os estudos empregando cultura celular em monocamada encontram limitações no que concerne às interações bidimensionais entre as células e experimentos utilizando animais, devido à elevada variabilidade, não conseguem predizer os resultados em humanos, comprometendo a sua relevância clínica. À vista disso, a cultura tridimensional de células (3D) utilizando um biomaterial fabricado para promover a proliferação e diferenciação celular tem sido utilizada para recriar a complexidade de um tecido normal, permitindo uma maior e complexa interação celular. Visando mimetizar o ambiente encontrado in vivo, este trabalho investiu no desenvolvimento de uma derme reconstituída (equivalente dérmico) in vitro utilizando como matriz biológica o colágeno, componente mais abundante da derme como suporte para os fibroblastos humanos, assim como na avaliação da fotobiomodulação com luz em 630 nm. Foi preparada uma esponja a partir do colágeno de serosa porcina 1,1% hidrolisado por 96 h. A caracterização do biomaterial foi realizada pela determinação da porosidade, do diâmetro dos poros, da absorção de fluidos e por ensaios de biocompatibilidade, uma vez que estes parâmetros são importantes para a proliferação e diferenciação celular na consequente formação do tecido in vitro. O biomaterial exibiu porosidade de 95,2%, com poros medianos de 44 μm estimados por porosimetria de injeção de mercúrio, além de canais com distância média entre as paredes de 78+/-14 μm estimado por MEV. Esses valores são considerados como ideais para um biosuporte de crescimento de fibroblastos. A absorção de água e meio de cultura foi de 95% e a esponja não apresentou citotoxicidade para a linhagem celular Vero. Adicionalmente, foi investigado o efeito de irradiação na cultura 3D com luz vermelha (dose 30 J/cm2), que mostrou fotobiomodulação na dose de 30 J/cm2 para cultura de células em monocamada e no início da fase de crescimento celular em cultura tridimensional. Por microscopia confocal, verificou-se que as células cultivadas na presença da esponja (cultura 3D), apresentaram diferenciação e secreção de matriz extracelular. Portanto, os resultados apresentados mostraram que a esponja de colágeno utilizada como biomaterial para suporte celular é eficiente para a produção de uma derme reconstituída (equivalente) in vitro e que a fotobiomodulação em 630 nm na dose de 30 J/cm2 de fato acelera o crescimento celular na matriz.The development of biomaterials substitutes and/or equivalents to mimic normal tissue is a currently challenge in tissue engineering. Studies using cell monolayer culture presents limitations with respect to two-dimensional interactions between the cells, and experiments using animals cannot predict results in humans, due to the high viability, thus compromising their clinical relevance. In consequence, three-dimensional cell culture (3D) using a biomaterial designed to promote cell proliferation and differentiation has been used to recreate the complexity of a normal tissue, allowing a larger and complex cellular interaction. Aiming to mimic the in vivo environment, the present work refers to create a reconstituted dermis (dermal equivalent) in vitro using collagen, the most abundant component of the dermis, as biological matrix, as support for human fibroblasts, as well evaluate the photobiomodulation with light at 630 nm. First, a sponge was prepared from serous 1.1% porcine collagen hydrolyzed for 96 h. The biomaterial was characterized by determination of its porosity, pore diameter, the fluid absorption and the biocompatibility assays, since these parameters are important to the cell proliferation and differentiation resulting in the in vitro tissue formation. The biomaterial showed porosity of 95.2%, with a median pore of 44 μM estimated by mercury porosimetry injection, and channels with an average distance between the walls of 78+/-14 μM estimated by SEM. These values are considered as ideal for a biosupport fibroblast growth. The absorption of water and growth medium was 95%, and the sponge showed no cytotoxicity for the Vero cell line. Additionally, it was investigated the effect of irradiation in 3D culture with red light (dose 30 J/cm2), that showed photobiomodulation on the dose 30 J/cm2, for culturing cells in monolayer and in the early-stage of the cell growth in three-dimensional culture. By confocal microscopy, it was verified that the cells cultured in the presence of the sponge (3D culture), allows differentiation and extracellular matrix secretion. Therefore, the results showed that the collagen sponge used as a biomaterial for cell support and the photobiomodulation at 630 nm and dose of 30 J/cm2 are efficient for the production of a reconstructed dermis (equivalent) in vitro.
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Hunter, Nikolas Ross. "In vitro studies on endothelial cells." Thesis, Heriot-Watt University, 1987. http://hdl.handle.net/10399/1040.
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Culard, Jean-François. "Etude des annexines de l'épiderme humain normal et reconstruit in vitro." Montpellier 1, 1992. http://www.theses.fr/1992MON11160.
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Massa, Denise Marie. "Is Organic Tissue Culture of Petunia Possible?" Available to subscribers only, 2009. http://proquest.umi.com/pqdweb?did=1796330171&sid=6&Fmt=2&clientId=1509&RQT=309&VName=PQD.
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Legrand, Claire. "Méthodes d'étude de la tolérance oculaire "in vivo" et "in vitro"." Paris 5, 1992. http://www.theses.fr/1992PA05P079.
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Tlili, Sham. "Biorhéologie in vitro : de la cellule au tissu." Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCC146.
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Nous avons développé au cours de cette thèse des expériences de rhéologie sur des tissus formés in vitro, qui sont des systèmes modèles pour étudier les propriétés mécaniques de tissus similaires aux tissus embryonnaires. Dans un premier temps, nous étudions l'origine microscopique de la viscosité effective d'agrégats cellulaires, qui sont des sphères auto-agrégées de cellules adhérentes. Pour cela, nous aspirons ces agrégats à travers des canaux microfluidiques afin que le tissu se déforme. Grâce à l'imagerie biphotonique, nous avons accès aux contours cellulaires, ce qui nous permet de quantifier les déformations et réarrangements cellulaires au cours de l'aspiration. Dans un second temps, nous étudions le mouvement collectif de monocouches cellulaires, qui sont des tissus bidimensionnels. Nous regardons comment est modifiée la migration collective d'une monocouche confinée dans une bande adhérente, lorsque les cellules rencontrent un obstacle non adhérent. L'étude du contournement de l'obstacle par le tissu nous renseigne sur les propriétés mécaniques de la monocouche. Dans les deux expériences décrites précédemment, les cellules subissent des gradients de vitesse importants et hétérogènes dus à la géométrie imposée du flot. L'analyse quantitative de ces expériences nous permet d'établir des analogies entre le comportement mécanique des tissus in vitro et celui d'un matériau cellulaire amorphe, tout en soulevant les différences dues à l'activité biologique. En parallèle de ces expériences, nous avons développé un formalisme qui intègre de façon cohérente des ingrédients mécaniques et biologiques rencontrés dans les tissus in vitro et in vivoWe have chosen in this thesis to perform rheology experiments on in vitro cell assemblies, which are established model systems to study the mechanics of embryonic-like tissues. First, we study the microscopic origin of the effective viscosity of cellular aggregates, that are three-dimensional self-assembled spheres of adherent cells. For that, we aspire aggregates through a narrow hole in a microfluidic channel. Using two-photon microscopy, we quantify the dynamics of individual cell deformation and cell rearrangements during the tissue deformation. Second, we study the collective movement of cell monolayers, that are bidimensional tissues. We look at how collective migration of a cell monolayer in a confined strip is disturbed by a circular obstacle placed in the middle of the strip. Quantifying the tissue flow and deformation around the obstacle gives insights on the monolayer mechanical properties. In these two experiments, cells undergo strong and heterogeneous velocity gradients due to set-up geometry. Quantitatively analyzing these experiments enables us to determine whether the mechanical behaviour of a cell assembly displays analogies with that of cellular materials known in physics, and what are its specificities directly related with cell activity. In parallel, we developed a mechanical formalism allowing to write constitutive mechanical equations in terms of intracellular and intercellular variables, that are the canonicat variables that we quantify in experiments. This formalism is a toolbox that enables to integrate consistently several mechanical and non-mechanical ingredients encountered in both in vitro and in vivo tissues
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Morris, Susan Debra. "Myocardial protection : from cell culture to human in vitro models." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298817.
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Konrad, Josef. "In vitro culture of Babesia divergens : biochemical and immunological investigations." Thesis, Imperial College London, 1986. http://hdl.handle.net/10044/1/38073.
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Lodge, Peter Graham. "In vitro culture of chicken and mouse embryo-derived cells." Thesis, University of Edinburgh, 2003. http://hdl.handle.net/1842/12455.
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Mouse embryonic stem (mES) cells can be maintained in vitro without loss of pluripotency in the presence of leukaemia inhibitory factor (LIF). Germline competent mES cells can be genetically modified in vitro and used to make transgenic mice via chimaeras. Germline competent chicken ES (chES) cells would be a powerful tool for the production of transgenic chickens. ES cells have been isolated from primates and mice but attempts from other species have been unsuccessful. Novel ES cell isolation strategies have been tested using inbred mouse strains. Using standard techniques, mES cells cannot be isolated from CBA strain embryos whereas they can be isolated from 129Sv strain embryos. A vital function of LIF in mES cells is activation of signal transducer and activator of transcription 3 (STAT3). LIF also activates the mitgoen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathway that appears to promote differentiation. LIF is a member of the interleukin-6 (IL-6) family of cytokines that includes ciliary neurotropic factor (CNTF). Manipulation of IL-6 family signals is a new approach to mES cell isolation that may be applied to development of methods of chES cell isolation. Prior to investigation of new approaches, standard mES cell isolation was performed. chES cells were not isolated in conditions adapted from standard mES cell isolation. A new approach involving the manipulation of LIF-mediated signals were evaluated in mES cell isolation experiments. The drug PD98059 inhibits the MEK/ERK pathway by preventing phosphorylation of MEK I. ES cell isolation frequency from strain 129Sv embryos increased significantly in the presence of 25μM PD89059 and 500 U/ml LIF (p<0.1) however, CBA ES cells could be isolated in the same conditions. The drug U0126 blocks and MEK/ERK pathway by directly inhibiting both phosphorylated MEK I and MEK II. CBA ES cells were isolated at a frequency of 22.3% in medium containing 2μM U0126 and 2x103 U/ml LIF.
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Yang, Jie. "Three dimensional perfused cell culture for in vitro toxicity testing." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:a72b7015-cc57-4bb8-904a-a5a88e2194f1.
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This study describes the development of a novel method of three dimensional perfused cell culture for in vitro toxicity testing. Multiple parallel perfused microbioreactors (TissueFlexTM) were adopted to provide a well-controlled cell culture environment. Alginate and collagen type I, commonly used as hydrogel scaffolds to support cell culture, were tested as the scaffolding materials for this application. Alginate supports cell proliferation, but does not support cell attachment. Collagen gel (type I), good for cell attachment but with poor mechanical strength, could be used at the high concentration of 5mg/ml to prevent the degradation of the gel. Improvement of collagen biomechanical property by a purpose-designed compressor to physically induce cross-linking showed promising results and merits further study. The suitability of alamarBlue® assay, a common non-toxic non-destructive viability assay method, was confirmed for this study and the protocol was optimised. To demonstrate the effectiveness of three dimensional perfused cell culture, human mesenchymal stem cells (MSC) seeded in collagen type I were employed to test the cell inhibition of two antibiotics, trimethoprim and pyrimethamine. The results displayed the perfusion system has greater advantage and sensitivity than the static system, as does these of 3D scaffolds, compared with 2D. Such differences are related to the continuous supply of fresh culture medium to keep cells at a stable pH, temperature, oxygen, and a more physiological like environment. The cytotoxicity of two stereoisomer compounds, obtained confidentially from Pfizer. Ltd., was assessed using the developed method and compared to conventional 2D static and perfused culture by using rat adipose mesenchymal stem cells. The results successfully distinguished toxic and non-toxic compounds and also demonstrated that the 3D perfused system improved the prediction of drug toxicity over 2D culture. 3D perfused bioreactors were applied to hepatotoxicity study using freshly isolated rat hepatocytes. Only algimatrixTM supported hepatocyte spheroid formation among those tested including collagen type I, alginate beads, poly lactic acid fibres, and AlgimatrixTM. A new variation of TissueFlexTM bioreactor with micro-patterned surface, designed specifically for hepatocyte self-assembly culture without use of any scaffold, was tested. The results demonstrated that, compared with the standard sandwich culture, the self-assembly culture in the micro-patterned bioreactors showed high cell viability, biomarkers expression, as well as more physiological immunocytochemistry. Moreover, the differential gene expression indicated that self-assembly culture could provide more relevant information regarding metabolising processes than the 2D sandwich culture, which would potentially improve hepatotoxicity prediction. In conclusion, 3D perfused cell culture for in vitro toxicity testing improved the predictivity, reliability and physiological relevance of drug toxicity compared to traditional 2D culture.
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Cook, James L. "Three-dimensional chondrocyte culture : in vitro and in vivo applications /." free to MU campus, to others for purchase, 1998. http://wwwlib.umi.com/cr/mo/fullcit?p9924877.
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Morselli, M. "THREE-DIMENSIONAL SCAFFOLDS FOR IN VITRO CULTURE OF FELINE OOCYTES." Doctoral thesis, Università degli Studi di Milano, 2016. http://hdl.handle.net/2434/367447.
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The cumulus-denuded oocytes (CDOs) are not commonly involved in the assisted reproductive techniques (ARTs), as the absence of their surrounding cumulus cells negatively affects their maturational and developmental competence in vitro.
In some cases these gametes could represent an interesting option for widening the germinal pool of high value individuals with very precious genetic material and could be the only genetic source when the selected cumulus-oocyte complexes (COCs, grade I) are cryopreserved.
Therefore, enriched culture conditions to improve the CDOs in vitro full competence should be adopted.
The innovative three-dimensional (3D) scaffolds, derived from the bioengineering and nanotechnology research, ensure the optimal culture conditions to maintain the cells physiological conformation and behavior as in the in vivo environment.
In this thesis, the association of 3D barium alginate microcapsules with competent COCs was used to improve the in vitro performances of domestic cat CDOs.
The results showed that the 3D BA microcapsules are suitable systems for the in vitro culture of feline oocytes, as their viability and in vitro maturation rates and embryonic development were similar to those obtain in the traditional 2D system.
The enriched co-culture condition did not improve the in vitro competence of CDOs, as their full maturational (TI-MII) rates and late embryo stages (morulae and blastocysts) were similar to those of the CDOs cultured separately or to those of the COCs control.
However, the presence of CDOs in the co-culture with COCs improved significantly the in vitro developmental rates of the high competence oocytes, presumably for the paracrine action of some specific oocyte-secreted factors (OSFs).
A better knowledge of the expression profiles of potential oocyte quality markers, as the OSFs, and how they could differ from COCs and CDOs in the 3D and 2D systems could help the design of the optimal enriched culture conditions for the domestic cat low competence oocytes.
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Pijut, Paula M. "Effects of culture filtrates of Ceratocystis ulmi on growth and ultrastructure of in vitro cultured Ulmus Americana /." The Ohio State University, 1988. http://rave.ohiolink.edu/etdc/view?acc_num=osu148759742413622.
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Lopes, Pedro Henrique Martins. "Estudos de Cultura de Tecidos, In Vitro, de Macroalgas Marinhas da EspÃcie Gracilaria birdiae." Universidade Federal do CearÃ, 2008. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=6571.
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nÃo hÃMacroalgas marinhas constituem um recurso vital para a economia de diversos paÃses e tem sido exploradas ao redor do globo devido a sua capacidade de produÃÃo de fico colÃides como o Ãgar, carragenina e alginatos, bem como pelo seu espectro de utilizaÃÃo, seja na indÃstria de alimentos, de fertilizantes e na medicina. Estudos sobre a induÃÃo, cultivo e reorganizaÃÃo de calos, tem representado um importante papel nas tÃcnicas de cultura de tecidos e suas aplicaÃÃes. Em diferentes tecidos cultivados in vitro, a utilizaÃÃo de reguladores de crescimento à de importÃncia primordial para o estabelecimento da competÃncia e determinaÃÃo, condiÃÃes necessÃrias para a formaÃÃo de calos e regeneraÃÃes. No presente estudo foram testados os efeitos de uma auxina e de uma citocinina, em diferentes concentraÃÃes de Ãgar em meio ASP 12-NTA. O efeito do Ãcido indolacÃtico (AIA) e da 6-benzilaminopurina (BAP) foram testados em explantes de macroalgas de espÃcie Gracilaria birdiae, em separado, com duas concentraÃÃes de 0,5 mg/L e 0,8 mg/L e em conjunto, nas concentraÃÃes de 1:5 mg/L e 5:1 mg/L. Com relaÃÃo a concentraÃÃo de Ãgar, foram testadas duas concentraÃÃes, com 0,5% e 0,8% para todos os tratamentos. Para o processo de esterilizaÃÃo foram aplicados um fungicida (nistatina) e um antibiÃtico (ciprofloxacina), alÃm de iodopovidona e hipoclorito de sÃdio. Todo o experimento foi conduzido em cÃmara de germinaÃÃo, com temperatura de 25  2oC e fotoperÃodo de 16hs de luz e 08hs escuro, salinidade de 30  2â e pH em torno de 7,6, durante aproximadamente 60 dias. Ao final de 50 dias de cultivo, foi observada a formaÃÃo de calos e de processos de regeneraÃÃo indireta dos mesmos. Os resultados foram submetidos a tratamentos estatÃsticos de anÃlise de contigÃncia atravÃs do Quiquadrado (χ2 ), onde foram observadas diferenÃas significativas entre a incidÃncia de calos e regeneraÃÃes e os nÃveis de Ãgar estudados. Ou seja, nos tratamentos onde foram utilizados nÃveis de Ãgar de 0,8%, apresentaram o maior nÃmero de regeneraÃÃes
Seaweed consists in a vital resource to the economy of many countries and it has been explored all over the world thanks to its capacity of producing colloids such as agar, carrageen and sodium alginate as well as the useful aspect either in food industry, fertilizers or medicine area. Studies on induction, cultivation and callus reorganization have been shown an important issue on tissue culture technique and its usage. In different tissue experience in vitro the usage of regulating growth is so fundamental to establish the competence and necessary conditions to callus formation and regeneration. In this present study has been tested the auxin and citocinin effects in different dosages of agar in ASP 12-NTA. The indolacetic acid effect and 6- benzilalaminopurine have been tested as well in seaweed in the species Gracilaria birdiae with two separated doses, 0,5mg/L and 0,8mg/L and together in concentration of 1:5mg/L and 5;1mg/L. Regarding to agar concentration, it has been tested two concentrations with 0,5% and 0,8% in all treatments. As to sterilization process has been done with fungicide (nistatina) and antibiotic (ciprofloxacin) besides iodopovidona and the sodium hypochlorite. The complete experiment had been lead in a germination chamber with 25Â2ÂC and photoperiod of more or less 16 hs of light and 08hs dark and 30,2â saltiness, pH around 7,6 within 60 days approximately. In more or less 50 days of cultivation had been noticed callus formation and regeneration process therein. The results had been underwent by statistic analyze treatments of contingent through (x2) where many meaningful different observations had been checked in callus and regeneration and the agar levels studied, that is to say the agar level treatments in 0,8% presented a major number of regeneration
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Ribeiro, Hugo Miguel Antunes. "Ensaios sobre microenxertia em nogueira (Juglans regia L.)." Master's thesis, Universidade de Évora, 2021. http://hdl.handle.net/10174/29912.
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As técnicas de cultura in vitro têm contribuído para a optimização de protocolos de propagação do género Juglans. Neste trabalho foi testada a técnica de microenxertia de J. regia em híbrido ‘Paradox’. Foi efectuado o enraizamento ex vitro em simultâneo com a enxertia. Foi avaliada a influência da presença de folhas no enxerto e no porta-enxerto na taxa de sucesso da técnica, assim como o despiste de Cherry leaf roll virus. Verificou-se que a presença de folhas foi um requisito obrigatório para o sucesso da enxertia e do enraizamento. A enxertia apresentou uma taxa média de sucesso de 84,6 % e a taxa de aclimatização aos 60 dias foi 86,4 %. Detectaram-se amostras suspeitas de serem positivas para a presença de Cherry leaf roll virus. A técnica de microenxertia apresentada mostrou-se viável tecnicamente e eventualmente com condições de competir com as técnicas tradicionais de enxertia em viveiro; ABSTRACT:
Trials about walnut (Juglans regia L.) micrografting.
In vitro culture techniques have contributed to the optimization of propagation protocols of the genus Juglans. Here the micrografting technique was tested with J. regia grafted in a 'Paradox' hybrid. Rooting ex vitro was performed simultaneously with grafting. The influence of the presence of leaves on the graft and rootstock on the success rate of the technique was evaluated, as well as the screening of Cherry leaf roll virus. It was found that the presence of leaves, in the graft or in the rootstock, or in both, was a mandatory requirement for the grafting success. The grafting success presented a final global average of 84.6% and the acclimatization rate at 60 days was 86.4%. Samples putatively positive for the presence of Cherry leaf roll virus were detected. The micrografting technique here presented seems to be technically feasible and maybe able to compete with the traditional techniques of nursery grafting.
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Rochd, Tarik. "Désinfection d'échantillons dentaires incubés in vitro avec des bactéries buccales." Toulouse 3, 1993. http://www.theses.fr/1993TOU30258.
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Le but de notre travail etait dans un premier temps d'etudier in vitro le comportement de 5 bacteries endocanalaires et parodontales (capnocytophaga ochracea, prevotella intermedia, peptostrep-tococcus micros, streptococcus mutans et streptococcus sanguis) vis a vis de dents bovines et dans un deuxieme temps de faire un criblage de 4 antimicrobiens (chlorhexidine, eugenol, chlorure de cetylpyridinium et hypochlorite de sodium) sur ces memes souches en suspension et en biofilm sur support dentaire. Des incisives de buf ont ete decoupees en petits cylindres de 3 mm de hauteur, nettoyees a l'hypochlorite de sodium, puis sterilisees et enfin incubees avec les bacteries en culture pure ou mixte. La colonisation de ces echantillons par les bacteries et leur migration a l'interieur des tubuli ont ete visualisees au microscope electronique a balayage, par des colorations histologiques pour s. Sanguis et par marquage immunofluorescent pour c. Ochracea. Du fait de sa mobilite, c. Ochracea apparait le plus apte a migrer a l'interieur des tubuli. Les autres bacteries presentent des potentialites d'envahissement variables. La colonisation des parois canalaire et cementaire et la migration a l'interieur des tubuli ont ete comparees a celles observees sur dents humaines extraites necrosees. Pour les biofilms mixtes, des coaggregations et des cotransports a faibles distances, de type piggy-back, ont ete observes entre certaines bacteries, notamment entre c. Ochracea et p. Intermedia. Dans un deuxieme temps, des solutions antiseptiques ont ete testees quant a leur capacite a desinfecter les echantillons dentinaires contamines. La chlorhexidine s'est montree la plus efficace a 0,10%, surtout en association avec l'eugenol (0,05%) et l'ethanol (7%). C. Ochracea s'est revele le plus resistant du fait certainement de sa propagation importante a l'interieur des tubuli (1342 micrometres)
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Travers, Albanne. "Congélation et maturation in vitro du tissu testiculaire prépubère de rongeurs." Rouen, 2013. http://www.theses.fr/2013ROUES051.
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Les cellules germinales souches à l‟origine de l‟élaboration des spermatozoïdes sont les cibles des thérapeutiques utilisés dans le traitement du cancer. Des stratégies de préservation des cellules germinales doivent être mises en place. Chez le jeune garçon, la congélation du tissu testiculaire et sa maturation ultérieure in vitro, évitant la réintroduction de cellules cancéreuses semblent être les stratégies les plus adaptées. Le travail présenté dans cette thèse a consisté en (i) l‟optimisation d‟un protocole de congélation du tissu testiculaire chez deux modèles animaux, la souris et le rat et (ii) l‟optimisation d‟un protocole de culture organotypique utilisant du rétinol et/ou de l‟acide rétinoïque, permettant d‟initier la spermatogenèse du tissu testiculaire décongelé de souris prépubères. L‟optimisation du protocole de congélation du tissu testiculaire prépubère de souris a montré que l‟utilisation de diméthylsulfoxyde (DMSO) à la concentration de 1,5M incubé pendant 30 minutes à 4°C et une courbe de descente en température contôlée lente sans « seeding » permet de préserver de façon optimale l‟architecture du tissu testiculaire de souris prépubère, de conserver la capacité proliférative des cellules germinales et des cellules de Sertoli et de maintenir l‟intégrité fonctionnelle du tissu testiculaire prépubère. L‟évaluation de différents protocoles de congélation du tissu testiculaire de rat prépubère, espèce présentant un déroulement de la spermatogenèse plus proche de celle de l‟homme par comparaison à la souris, confirme le protocole de congélation précédemment défini chez la souris et permet de préciser le poids du fragment à congeler (7,5mg) et le conditionnement de l‟échantillon (cryotubes), souhaitables pour une conservation optimale du tissu testiculaire prépubère. La culture organotypique de tissu testiculaire frais et décongelé de souris prépubère montre une prolifération des cellules intratubulaires, une croissance du tissu et une initiation de la spermatogenèse avec l‟utilisation de rétinol à la concentration de 10-6M similaires entre le tissu frais et le tissu préalablement congelé avec une phase de stabilisation de la température à −8°C. L‟intégrité fonctionnelle des cellules de Leydig est également correctement préservée puisqu‟une stéroïdogenèse in vitro est observée pour les deux types de culture. Ce travail a donc permis de confirmer et d‟affiner le protocole de congélation optimal pour la préservation de l‟intégrité fonctionnelle et structurale du tissu testiculaire prépubère et peut donc être proposé dans le cadre d‟une application humaine de préservation de la fertilité. Néanmoins, la maturation in vitro doit encore être optimisée pour obtenir une spermatogenèse complète. Il sera alors possible d‟envisager une utilisation sur le tissu testiculaire humain prépubère décongelé et ainsi de proposer une solution durable de préservation de la fertilité des garçons prépubères atteints d'un cancer, et d‟apporter des réponses aux questions posées par les patients sur la vie après le cancer et la prise en charge des complications et des séquelles induites par les traitements
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Cordeiro, Lívia da Silva. "Produção in vitro e criopreservação de raízes de Cleome rosea Vahl (Capparaceae)." Universidade do Estado do Rio de Janeiro, 2011. http://www.bdtd.uerj.br/tde_busca/arquivo.php?codArquivo=2410.
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Fundação de Amparo à Pesquisa do Estado do Rio de JaneiroCleome rosea é uma espécie nativa, de porte herbáceo, ocorrente em restingas brasileiras. Estudos recentes têm revelado o potencial medicinal da espécie para importantes propriedades farmacológicas, como por exemplo, as atividades anti-inflamatória, antigenotóxica, antiviral e antibacteriana. Porém, nos últimos anos, C. rosea não tem sido encontrada em várias regiões de seu ambiente natural, devido, principalmente, às ações antrópicas. Dessa forma, torna-se relevante o desenvolvimento de métodos de conservação que permitam o estudo e exploração das propriedades medicinais da espécie. O cultivo in vitro de raízes representa uma forma eficiente para produção de biomassa, devido ao rápido crescimento, produção estável de metabólitos, além de representar uma potencial fonte de explantes para a propagação em massa de diferentes espécies. O presente trabalho teve como objetivo a produção in vitro de culturas de raízes de C. rosea, associada à criopreservação, como forma de manutenção em longo prazo das culturas, monitorada através da análise de estabilidade genética. As culturas estabelecidas a partir de explantes radiculares de plantas propagadas in vitro de C. rosea demonstraram excelente capacidade de multiplicação de raízes em meio de cultura suplementado com o fitorregulador ANA, com manutenção dessa capacidade ao longo de sucessivas subculturas. Associado a esses resultados, o estabelecimento de protocolos de criopreservação pelo método de vitrificação resultou em elevados valores de frequência de recuperação do material após congelamento em nitrogênio líquido com as soluções de vitrificação PVS2 e PVS3. Os estudos de monitoramento da estabilidade genética, pela técnica de marcadores moleculares RAPD, revelaram a presença de polimorfismos significativos em uma das três culturas iniciadas a partir de raízes de C. rosea criopreservadas. Esses resultados demonstram as possibilidades de produção de raízes de C. rosea e conservação em longo prazo através da criopreservação, iniciando estudos inéditos para a espécie.
Cleome rosea is an herbaceous species found in the restinga vegetation of Brazil. Recent studies have reported its medicinal potential for important pharmacological properties, such as anti-inflammatory, antigenotoxic, antiviral and antibacterial activities. However, in recent years, no C. rosea plants have been found in their natural habitat, mainly due to human impact. Thus, it becomes applicable to the development of conservation methods that facilitate the study and exploration of the medicinal properties of C. rosea. The in vitro roots culture represents an efficient way to produce biomass, due to rapid growth, stable production of metabolites and represent a potential source of explants for mass propagation of different species. This study aimed to produce in vitro root cultures of C. rosea, associated with cryopreservation, as form of long-term maintenance of cultures, monitored by analysis of genetic stability. Cultures established from root explants of in vitro propagated plants of C. rosea showed excellent ability to multiplication of roots in culture medium supplemented with NAA plant regulator, maintaining this capacity during successive subcultures. Associated with these results, the establishment of protocols for cryopreservation by vitrification method resulted in high values of recovery frequency of the material after freezing in liquid nitrogen with vitrification solutions PVS2 and PVS3. Studies to monitoring the genetic stability by RAPD technique revealed the presence of significant polymorphisms in one of three cultures initiated from cryopreserved roots of C. rosea. These results demonstrate the potential production of roots of Cleome rosea and long-term conservation through cryopreservation, starting unpublished studies for the species.
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Vebret, Laurence. "Production in vitro de polymères extracellulaires par la cyanobactérie thermophile Mastigocladus laminosus." Limoges, 1999. http://www.theses.fr/1999LIMO0026.
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Connues pour leur capacite a produire des polymeres extracellulaires et a s'adapter a des milieux tres divers, les cyanobacteries semblent etre une source interessante pour des biopolymeres ayant des proprietes physico-chimiques, rheologiques, cosmetologiques, therapeutiques et antimicrobiennnes. Aussi, une etude in vitro a ete realisee sur mastigocladus laminosus, une cyanobacterie thermophile isolee a partir de la biomasse thermale de neris-les-bains, afin de mieux connaitre les conditions culturales favorables au developpement de ce micro-organisme et a sa production de polymeres extracellulaires. Par consequent, differents parametres ont ete testes tels que des facteurs physiques : l'intensite lumineuse, la temperature, la photoperiode, la taille du bioreacteur ; physico-chimiques : le ph ; ou chimiques : la composition du milieu de culture (dilutions du milieu de culture de reference et concentration en bicarbonate). La production de biomasse seche et de polymeres extracellulaires ainsi que la composition de ces derniers et le contenu pigmentaire de m. Laminosus sont determines pour evaluer l'influence des differentes conditions de culture in vitro. Contre toute attente, ce ne sont pas les facteurs physiques telles que l'intensite lumineuse et la temperature (excepte pour les conditions extremes) qui font varier le plus le developpement de m. Laminosus et sa production en polymeres extracellulaires et leur nature mais essentiellement la composition du milieu de culture. Cette etude a permis de recueillir de precieuses indications pour l'amelioration de la production de biomasse et de polymeres extracellulaires en vue d'une valorisation d'une biomasse thermale.
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Mahon, François-Xavier. "L'élimination in vitro des cellules leucémiques de la leucémie myéloi͏̈de chronique." Bordeaux 2, 1997. http://www.theses.fr/1997BOR28474.
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Kalman, Benoît. "Génération et optimisation de microtissus musculaires 3D in vitro." Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAI053.
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L’ingénierie du tissu musculaire squelettique vise à reconstituer in vitro un tissu fonctionnel aussi physiologique que possible dans le but de mieux comprendre la myogenèse, l’impact de mutations génétiques et tester des médicaments. Ces dernières années, différents modèles de tissus musculaires tridimensionnels ont été développés. Toutefois, l’utilisation prépondérante de cellules murines et la taille de ces modèles restreint leur pertinence pour les études de pathologies humaines et le criblage pharmacologique. Dans le cadre de ce travail de thèse, nous avons donc développé différents modèles de tissus musculaires humains micrométriques pour répondre à ces limitations. Dans un premier temps, nous avons conçu et optimisé par microfabrication une plateforme caractérisée par la présence de microcanaux. Nous avons ainsi généré des tissus musculaires multicouches alignés présentant une organisation proche du muscle natif à partir de myoblastes murins immortalisés C2C12 puis de myoblastes humains immortalisés. Nous avons ainsi montré l’influence de la topographie et de la concentration cellulaire sur l’alignement des myotubes et la maturation du tissu musculaire. Dans un second temps, nous avons développé une plateforme constituée de micropuits contenant chacun deux micropiliers permettant d’analyser la contractilité des tissus. Des microtissus musculaires 3D standardisés ont ainsi été générés avec cette plateforme à partir de myoblastes murins, et de myoblastes C2C12 électroporés avec un gène muté ou non de la desmine. Par la suite, des microtissus ont été générés à partir de myoblastes humains. L’importance du choix de la matrice dans la formation des microtissus et les bénéfices d’une coculture de myoblastes et fibroblastes dans la stabilité des tissus ont ainsi été mis en évidence. La géométrie de micropiliers a aussi été optimisée afin de générer et comparer des microtissus composés de myoblastes isolés de patients sains et malades (dystrophie musculaire de Duchenne). Une preuve de concept démontrant la possibilité d’utiliser cette technologie pour tester des thérapies chimiques et géniques a été établie. Nous avons en effet suivi en temps réel les effets de l’inhibiteur de la kinase Rho-associée Y-27632 sur la contractilité des microtissus, ainsi que la transduction d’un gène rapporteur fluorescent modèle par les cellules composant les microtissus. Les résultats de ce travail de thèse démontrent le potentiel de cette technologie pour l’étude des processus fondamentaux de la myogenèse, l’évaluation des effets fonctionnels de mutations patient-spécifique et le criblage de thérapies chimiques et géniquesSkeletal muscle tissue engineering aims to build functional and physiological tissues in vitro in order to better understand myogenesis, to investigate the impact of genetic mutations and to screen potential therapies. Over the past few years, bi- and tridimensional models of muscle tissue have been developed, but most of these models are based on the use of murine cells and require large amounts of cells, thus limiting their relevance to study pathologies of human muscles and drug screening assays. Here we aimed at developing different models of human muscle microtissues to address these issues. By using microfabrication techniques, we first engineered a microgrooved platform we used to generate aligned multilayered skeletal muscle tissues from murine C2C12 myoblasts and human immortalized myoblasts. We showed the impact of topography and cell density on the maturation and myotube alignment. We then fabricated a microdevice, consisting of microwells containing two micropillars allowing an easy access to the contractility of muscle tissues. We engineered microtissues from C2C12 and C2C12 myoblasts electroporated with a mutated gene of desmin, and showed some limitation of this technique of transduction. Finally, we generated microtissues from human myoblasts. We investigated the role of the extracellular matrix in the tissue formation and evidenced the benefits of coculturing myoblasts and fibroblasts on the stability of muscle microtissues. Furthermore, we optimized the geometry of the micropillars to engineer and compare microtissues composed of human myoblasts isolated from healthy and diseased (Duchenne muscular dystrophy) patients. A proof of concept of the potential of this technology for screening chemical and gene therapies was established. We were indeed able to analyze in real time the effects of the Rho-associated kinase-inhibitor Y-27632 on the tissue contractility, as well as the transduction of a model fluorescent reporter gene. Altogether, the results of this work demonstrate the potential of this technology to study fundamental muscle biology, examine functional effects of patient-specific mutations or screen chemical and gene therapies
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Straub, Volko A. "In vitro study of a central pattern generator." Thesis, University of Sussex, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.285209.
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Ndeberi, Jean. "Induction de l'organogenèse chez Oryza sativa par la culture in vitro." Doctoral thesis, Universite Libre de Bruxelles, 1985. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/213625.
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Ling, Shanhong. "Effects of estrogens on the vasculature in vitro cell culture studies." Monash University, Dept. of Medicine, 2003. http://arrow.monash.edu.au/hdl/1959.1/9345.
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Châteauneuf, Isabel. "Culture in vitro de lymphocytes B humains, aspects cellulaires et moléculaires." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ43799.pdf.
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Sturgeon, Tracey Eileen. "The in vitro culture and regeneration of elite Canadian barley genotypes." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/MQ57584.pdf.
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Peters-Hall, Jennifer Ruth. "In Vitro Cell Culture Models to Study Cystic Fibrosis Respiratory Secretions." Thesis, The George Washington University, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=3597271.
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Cystic fibrosis (CF) is the most common lethal autosomal recessive genetic disorder that affects the Caucasian population. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR), and is characterized by a viscous airway surface liquid (ASL) that impairs mucociliary function and facilitates bacterial infection. The molecular mechanisms by which these symptoms result from CFTR malfunction are unclear. We hypothesized that expression and secretion of innate immune proteins is altered in CF ASL.
We sought to use cell culture models in which the only source of secreted proteins was differentiated airway epithelium. Since CFTR localizes to the apical surface of airway submucosal glands (SMG) and ciliated epithelium, cell culture models that recapitulate two parts of respiratory tract epithelium were studied: 1) SMG acini and 2) mucociliary epithelium.
We developed a three-dimensional system wherein CF (ΔF508/ΔF508) and non-CF human bronchial epithelial (HBE) cells differentiated on Matrigel into polarized glandular acini with mature lumens by two weeks with no significant variability in size. Bronchial acini expressed and secreted SMG proteins, MUC5B and lysozyme, at day 22, and exhibited vectorial secretions that were collected along with acinar cell lysates. Proteome profiling demonstrated unique protein signatures for each cellular space. However, abundant contaminating proteins from Matrigel and growth media were identified. Therefore, the ALI cell culture model of airway epithelium was chosen for quantitative proteomic comparison of CF and non-CF HBE apical secretions because the protein-rich media does not contact the apical surface.
CF and non-CF HBE cells were labeled by stable isotope labeling with amino acids in cell culture and differentiated at ALI. LC-MS/MS and bioinformatic analysis identified seventy-one proteins with altered levels in CF secretions (+/−1.5 fold-change; p-value<0.05). Validation with antibody based biochemical assays demonstrated increased levels of MUC5AC, MUC5B, fibronectin and MMP9, and increased proteolysis/activation of complement C3, in CF secretions. Overall, the function of altered proteins in the CF secretome is indicative of an airway epithelium in a state of repair and altered immunity in the absence of infection, suggesting the downstream consequences of mutated CFTR in CF airways set the stage for chronic inflammation and infection.
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Zeyfert, Caroline Margaret. "Surface functionalised emulsion-templated porous polymers for in-vitro cell culture." Thesis, Durham University, 2010. http://etheses.dur.ac.uk/794/.
Full textAbstract:
“PolyHIPE” is an acronym for polymerized high internal phase emulsions. The nature of the formation of PolyHIPEs creates a highly porous, interconnected monolith structure, the architecture of which can be tightly controlled. Styrene-2-ethylhexylacrylate-divinylbenzene PolyHIPEs with defined architecture of voids between 80 – 100 μm have been previously investigated as suitable supports for in-vitro cell culture, but the highly hydrophobic nature of the predominantly polystyrene scaffold requires extra processing steps to hydrate the surface before use as a support for cell culture. This thesis addresses routes to surface functionalise these PolyHIPEs for the specific aim of optimising 3D in-vitro cell culture materials. Specific routes to this include chemical modification, plasma treatment and chemical adsorption. Of these three routes to surface functionalisation, the plasma processing appears to give the best results, with further attachment of biologically-directing molecules, possible. This thesis presents oxygen plasma treatment as a route to increase the hydrophilicity of these materials, with a reasonable shelf-life, which both reduces the processing steps before cell culture, and increases cell viability when grown on the functionalised PolyHIPE. The ultimate aim in this project is to create smart “off-the-shelf” materials that can control cell behaviour in-vitro. Chemical attachment to the surface of the PolyHIPE with synthetic retinoid EC23 has been proposed, and initial chemical tests obtained to suggest attachment, with future testing with mammalian cells envisaged.
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Dumont, Nellie. "Production d'anticorps spécifiques in vitro par culture de lymphocytes B humains." Thesis, Université Laval, 2008. http://www.theses.ulaval.ca/2008/25130/25130.pdf.
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Sassano, Emily. "Role of regulatory T cells in in vitro human culture systems." Honors in the Major Thesis, University of Central Florida, 2007. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/1046.
Full textAbstract:
This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.Bachelors
Burnett College of Biomedical Sciences
Molecular and Microbiology