Dissertationen zum Thema „Cell coculture“
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1
Apple, Allon Aliza. „Bilaminar coculture of stem cells and instructive cells for tissue regeneration“. Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3390115.
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Thesis (Ph.D.)--University of California, San Francisco with the University of California, Berkeley, 2009.Source: Dissertation Abstracts International, Volume: 71-02, Section: B, page: . Adviser: Jeffrey C. Lotz.
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Hakelius, Malin. „Interactions between Malignant Keratinocytes and Fibroblasts : Studies in Head and Neck Squamous Cell Carcinoma“. Doctoral thesis, Uppsala universitet, Plastikkirurgi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-221109.
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Carcinoma growth requires a supportive tumor stroma. The concept of reciprocal interactions between tumor and stromal cells has become widely acknowledged and the connective tissue activation seen in the malignant process has been likened to that of a healing wound. Little is, however, known about the specific characteristics of these interactions, distinguishing them from the interplay occurring between epithelial and stromal cells in wound healing. In order to study differences in the humoral effects of malignant and benign epithelial cells on fibroblasts, we used an in vitro coculture model with human oral squamous cell carcinoma cells (SCC) or normal oral keratinocytes (NOK) on one side of a semi-permeable membrane and fibroblasts seeded in gels on the other. Pro-collagens α1(I) and α1(III) were more downregulated in NOK cocultures compared to SCC cocultures. IL-1α was identified as a major keratinocyte-derived soluble factor behind the effects observed. We concluded that SCC are less antifibrotic compared to NOK. There was also a differential expression among enzymes involved in ECM turnover. The urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) were both upregulated by NOK, but not by SCC. Here, rIL-1ra caused further upregulation of PAI-1. Global gene expression in fibroblasts was assessed using Affymetrix™ arrays. In total, 82 transcripts were considered differentially expressed; 52 were up- and 30 were downregulated in SCC compared to NOK cocultures. Among the differentially expressed genes there was an enrichment of genes related to collagens and to a nonspecific, innate-type response. The innate response marker pentraxin (PTX3) was upregulated by keratinocyte-derrived IL-1α in both NOK and SCC cocultures. We observed a considerably higher IL-1α / IL-1ra quotient in SCC cocultures, however, while PTX3 mRNA upregulation was higher in SCC cocultures, there was no difference in the level of PTX3 secreted protein. Taken together, we concluded that NOK and SCC regulate genes important for ECM composition and for the innate immune-response differentially. IL-1α was identified as one important mediator of the observed effects. In general, SCC appeared to be more profibrotic in their effects on fibroblasts.
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Chamayou, Léo. „LiverPearls, une méthode de culture multicellulaire miniaturisée et à haut débit reproduisant l’environnement physiologique et la structure tridimensionnelle du foie humain“. Thesis, Université Paris sciences et lettres, 2020. http://www.theses.fr/2020UPSLS005.
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L’intérêt pour de nouveaux modèles de foie, plus proches physiologiquement du foie in vivo, est élevé, en particulier en provenance de l’industrie pharmaceutique. En effet, les systèmes standards, tels que la culture en 2D ne sont pas très prédictifs pour certaines études et de meilleurs modèles sont nécessaires, à la fois dans le cadre des études ADME/Tox pour le développement de médicaments ou pour modéliser les nombreuses maladies hépatiques. Afin de reproduire le foie humain, un modèle doit imiter sa structure de façon plus proche que les systèmes en 2D et refléter sa composition cellulaire. Pour produire ce modèle, nous avons utilisé une technologie de micro-encapsulation basée sur la co-extrusion dans l’air d’un jet biphasique, composé d’une phase externe de solution d’alginate et d’une phase interne contenant les cellules. Ce jet est ensuite fragmenté puis l’alginate est réticulé produisant des micro-capsules cœur/coque. La coque poreuse d’alginate protège les cellules du stress mécanique tout en permettant le passage de l’oxygène et des nutriments.Les cellules s’auto-assemblent dans ces capsules en sphéroïdes hépatiques pouvant être cultivés pendant un mois en gardant une bonne fonctionnalité et pouvant être utilisés dans le cadre de criblage à haut-débit. Cette thèse a porté sur l’utilisation de cette technologie pour mettre au point un modèle 3D de foie humain contenant des hépatocytes primaires humains, des cellules de Kupffer, et des cellules endothéliales sinusoïdales. Dans un premier temps, les conditions de culture de ce modèle ont dû être optimisées, notamment le ratio entre les différents types cellulaires et le milieu de culture adapté à ceux-ci. Puis, une fois ces conditions établies, le modèle a été caractérisé, structurellement par microscopie,ainsi que fonctionnellement, par l’étude de l’expression génique de plusieurs protéines hépatiques importantes, telles que les cytochromes P450 ou des récepteurs nucléaires. Des études d’activité enzymatique, de sécrétion d’albumine et d’urée ont également été menées. Ces capsules nous permettent d’obtenir un modèle en 3D, plus proche de la structure du foie humain, et capable de reproduire les interactions complexes entre les différents types cellulairesInterest for new and more physiologically relevant liver models is high, particularly from pharmaceutical companies. Standard systems, like 2D culture, are indeed not enough predictive and better models are needed, either for drug candidates screening in ADME/Tox studies or for hepatic diseases modelling. To be closer to the human liver, a new model needs toreplicate liver structure and cellular composition better than the 2D. To this end, we used a micro-encapsulation technology, developed by the laboratory and based on the co-extrusion of a two-phases jet, composed of an alginate external phase and a cell-containing internal phase. This jet is then fragmented into micro-droplets and the alginate reticulated to form core-shell microcapsules. The porous alginate shell protects the cells from shear stress while letting oxygen and nutrients pass, and by preventing cell adhesion, enables the cells to self-assemble into hepatic spheroids which can bekept alive during one month, retain good functionality and can be used for high-throughput screening. This thesis focusedon using this technology to develop a next 3D liver model containing human primary hepatocytes, Kupffer cells and liver sinusoidal endothelial cells. Firstly, culture conditions for this model had to be optimized, particularly the ratio between these different cell types and the culture medium, which had to be suitable for these cell types. Then, once the culture conditions had been established, the model was characterized, structurally by immunofluorescence staining, and functionally by studying gene expression of important liver proteins, like cytochromes P450 or nuclear receptors. Enzymaticactivity, albumin and urea secretion were also studied. These capsules allow us to obtain a model able to replicate the complex interactions between these cell types and structurally closer to the human liver
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Messelmani, Taha. „Development and characterisation of a biomimetic liver on chip featuring 3D hepatic coculture with an endothelial barrier“. Electronic Thesis or Diss., Compiègne, 2023. http://www.theses.fr/2023COMP2736.
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Au cours des programmes de développement de médicaments, des modèles animaux sont utilisés pour évaluer le métabolisme et la toxicité des médicaments. Plusieurs cadres juridiques sont établis pour le remplacement, la réduction et l'amélioration de ces expériences. Le foie est un organe central pour la détoxification des molécules exogènes. Par conséquent, le développement de modèles reproduisant les fonctions du foie reste un objectif ambitieux. Ces dernières années, l'association entre l'ingénierie tissulaire et la technologie des organes sur puce a conduit au développement de modèles alternatifs imitant certaines fonctions hépatiques. L'objectif de ce travail est de développer une plateforme de foie sur puce biomimétique en couplant une biopuce d'hépatocytes et une barrière endothéliale. Dans la première partie, nous avons utilisé la technologie des organes sur puce et un hydroscaffold à base de matrice extracellulaire pour organiser les cellules en 3D. Les sphéroïdes formés ont été caractérisés sur le plan structurel et fonctionnel. Dans la deuxième partie, nous avons caractérisé la formation d'une barrière endothéliale. Nous avons établi les conditions de co-culture et analysé le potentiel du couplage de la barrière endothéliale avec la puce d'hépatocytes pour métaboliser l'APAP. Enfin, nous avons analysé la signature métabolomique de chaque condition, les interactions entre les cellules et identifié la signature métabolique des lésions causées par l'APAP. Dans la dernière partie, nous avons proposé des pistes d'amélioration en utilisant des hépatocytes primaires ou en intégrant la barrière endothéliale et les hépatocytes dans une biopuce bi-comportementaliséeDuring drugs development programs, animal models are commonly used for the assessment of the metabolism and toxicity of drug candidates. Several legal frameworks are being settled to promote the replacement, the reduction, and the refinement of these experiments. The liver is a central organ involved in the detoxification of exogenous molecules. Accordingly, the development of models mimicking the functions of the liver remain a challenging objective. Conventionally, liver cells are cultured in vitro in 2D Petri dishes but this conformation leads to a rapid loss of their functions. In recent years, the association between tissue engineering and organ-on-chip technology led to the development of more accurate alternative models that mimic the liver functions. The aim of this thesis is to develop a biomimetic liver-on-chip platform by coupling a hepatocyte biochip and an endothelial-like barrier. The goal is to mimic the passage of molecules through the liver sinusoid endothelial barrier and then their metabolism with the hepatocytes. In the first part, we used organ-on-chip technology and ECM-based hydroscaffold to organise the cells in 3D structures. The potential of our model was compared with static Petri dishes and the spheroids formed were characterised structurally and functionally. In the second part, we characterized the formation of an endothelial barrier and identified specific markers indicating the conservation of the phenotype of endothelial cells. We established the coculture conditions and analysed the potential of coupling the endothelial barrier with the hepatocyte-on-chip to metabolize the APAP as a candidate molecule. Finally, we analysed the metabolomic signature of each condition, crosstalk between the cells, and identified the metabolic signature of APAP injury and described the reactions happening at metabolic level. In the last part, we proposed tracks of improvement by using primary hepatocytes or by integrating the endothelial barrier and the hepatocytes in the same bi-compartmentalized biochip
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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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Joshi, Ramila Joshi. „Micro-engineering of embryonic stem cells niche to regulate neural cell differentiation“. University of Akron / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=akron1544029342969082.
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Madiedo-Podvršan, Sabrina. „Development of a lung-liver in vitro coculture model for the risk assessment of inhaled xenobiotics“. Electronic Thesis or Diss., Compiègne, 2022. http://www.theses.fr/2022COMP2703.
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L’urbanisation et la mondialisation sont des phénomènes de société qui multiplient et complexifient les sources de pollution. Parmi elles, la pollution atmosphérique impacte notablement la santé humaine à l’échelle mondiale de par son caractère transfrontière. L’appareil respiratoire est une voie d’absorption de nombreux xénobiotiques, sous forme de gaz, d’aérosols ou de nanoparticules. Une fois dans les voies respiratoires, les substances inhalées sont susceptibles d’interagir avec les cellules pulmonaires. Les mécanismes par lesquels des xénobiotiques inhalés induisent des dommages pulmonaires sont complexes, notamment en raison de l’hétérogénéité cellulaire des poumons. En raison de cette complexité, les modèles animaux constituent un outil de référence pour les études toxicologiques prédictives, cependant, dans le contexte européen de réduction de l’expérimentation animale (REACH, et les règles 3R), le développement de méthodes alternatives fiables est devenu une nécessité. Les modèles in vitro sont de bons candidats car plus simple et moins couteux à mettre en oeuvre que les modèles vivo et permettent de travailler avec des cellules ou des tissus d’origine humaine ce qui contribue à améliorer la pertinence des résultats. Cependant, l’extrapolation limitée du vitro au vivo est souvent liée à un manque de complexité des modèles, notamment en raison de l’absence de communication inter-organes. Les technologies des multi-organes sur puce cherchent à surmonter ces limitations en connectant plusieurs organoïdes métaboliquement actifs au sein d’un même circuit de culture afin de reproduire des interactions de type systémiques. Dans ce contexte, nous décrivons un modèle permettant de connecter in vitro, par le biais de la microfluidique, une barrière pulmonaire (voie d’entrée des xénobiotiques inhalés) à un organe détoxifiant tel que le foie, afin d’évaluer la toxicité liée à un stress inhalatoire de façon plus systémique. Cette approche permet de considérer la biotransformation des composés inhalés et l’interaction inter-organes comme possible modulateurs de la toxicité. Le projet étant dans les premières phase de développement, la robustesse expérimentale était au coeur du projet. L’objectif principal était de prouver qu’une substance modèle était capable de transiter dans le dispositif, au travers des deux compartiments tissulaires, afin de pouvoir étudier la dynamique inter-organes poumon/foie en condition de stress xénobiotique. Le projet a été articulé en trois phases expérimentales : - Caractérisation des réponses biologiques spécifiques aux tissus pulmonaire et hépatique en réponse à un stress. La viabilité, la fonctionnalité et les activités métaboliques des monocultures ont été évaluées après exposition à une substance modèle. - Adaptation et préparation des monocultures aux conditions de co-culture afin de préserver la viabilité et la fonctionnalité des tissus. - Les compartiments pulmonaire et hépatique ont été cultivés jointement dans un circuit de culture microfluidique fermé. La co-culture a été exposée à une substance modèle à travers la barrière pulmonaire afin d’imiter un mode d’exposition inhalatoire. Les paramètres de viabilité et de fonctionnalité des tissus ont été évalué post-culture afin de mettre en évidence quelconque phénomène d’interaction inter-organe. La caractérisation du modèle de co-culture a été réalisé grâce à l’exposition d’un agent hépatotoxique de référence, largement étudié dans la littérature : l’acétaminophène aussi connu sous le nom de paracétamol (APAP). L’exposition à la barrière pulmonaire n’est pas physiologique mais permet d’observer quantitativement le passage et la circulation du xénobiotique à travers le dispositif car l’APAP interfère avec la viabilité et les performances métaboliques hépatique, permettant ainsi de vérifier que le compartiment hépatique peut avoir accès à l’exposition effectuée à travers la barrière pulmonaireUrbanization and globalization are prevailing social phenomena that multiply and complexify the sources of modern pollution. Amongst others, air pollution has been recognized as an omnipresent life-threatening hazard, comprising a wide range of toxic airborne xenobiotics that expose man to acute and chronic threats. The defense mechanisms involved in hazardous exposure responses are complex and comprise local and systemic biological pathways. Due to this complexity, animal models are considered prime study models. However, in light of animal experimentation reduction (3Rs), we developed and investigated an alternative in vitro method to study systemic-like responses to inhalationlike exposures. In this context, a coculture platform was established to emulate interorgan crosstalks between the pulmonary barrier, which constitutes the route of entry of inhaled compounds, and the liver, which plays a major role in xenobiotic metabolism. Both compartments respectively comprised a Calu-3 insert and a HepG2/C3A biochip which were jointly cultured in a dynamically-stimulated environment for 72 hours. The present model was characterized using acetaminophen (APAP), a well-documented hepatotoxicant, to visibly assess the passage and circulation of a xenobiotic through the device. Two kinds of models were developed: (1) the developmental model allowed for the technical setup of the coculture, and (2) the physiological-like model better approximates a vivo environment. Based on viability, and functionality parameters the developmental model showed that the Calu-3 bronchial barrier and the HepG2/C3A biochip can successfully be maintained viable and function in a dynamic coculture setting for 3 days. In a stress-induced environment, present results reported that the coculture model emulated active and functional in vitro crosstalk that seemingly was responsive to high (1.5 and 3 mM) and low (12 and 24 μM) xenobiotic exposure doses. Lung/liver crosstalk induced modulation of stress response dynamics, delaying cytotoxicity, proving that APAP fate, biological behaviors and cellular stress responses were modulated in a broader systemic-like environment
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Kletting, Stephanie [Verfasser], und Claus-Michael [Akademischer Betreuer] Lehr. „A new cell line-based coculture model of the human air-blood barrier to evaluate the interaction with aerosolized drug carriers / Stephanie Kletting ; Betreuer: Claus-Michael Lehr“. Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2016. http://d-nb.info/1114735035/34.
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Forte, Andresa. „Expansão ex vivo das células-tronco hematopoiéticas do sangue do cordão umbilical: análise comparativa da proliferação celular em cocultura de células-troco mesenquimais provenientes do endotélio vascular do cordão umbilical e do tecido adiposo“. Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5167/tde-25022015-085731/.
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INTRODUÇÃO: As células-tronco hematopoiéticas (CTH) do sangue do cordão umbilical (SCU) têm sido utilizadas com sucesso para o tratamento de doenças malignas e não malignas. No entanto, algumas unidades de SCU podem apresentar baixa quantidade de células nucleadas totais (CNT). Algumas abordagens têm sido sugeridas para evitar problemas em relação à baixa concentração de CTH no transplante, como a administração de duas unidades de SCU para o paciente e a expansão ex vivo de CTH. OBJETIVO: Avaliar as taxas de proliferação celular na expansão ex vivo do SCU em sistema de cocultura com células-tronco mesenquimais (CTM) obtidos a partir de diferentes fontes com alta e baixa confluência e adicionando-se ou não coquetel de citocinas no meio de cultura. MÉTODOS: Este estudo foi aprovado pelo Comitê de Ética de Pesquisa (CAPPesq) do Hospital das Clínicas da Faculdade de Medicina da USP. A coleta do SCU (n =10) foi realizada após o nascimento do bebê e expulsão da placenta. O processamento foi realizado utilizando o método de redução de volume, o qual consiste em depleção de eritrócitos. As amostras de CTM provenientes do endotélio vascular do cordão umbilical foram obtidas de doadores diferentes (n=3) e o tecido adiposo (n=3) do inventário do LIM-31. A expansão das CNT e das células com expressão de marcadores CD133+/CD34+ foram observados depois de sete dias de cultura. Além disso, o ensaio para análise de unidades de formadoras de colônias (UFC) foi realizado em todas as amostras antes e depois da expansão do SCU. Para a expansão em sistema de cocultura foi separado dois grupos para ambas as fontes de CTM (Grupo I - cocultura com adição de coquetel de citocinas vs. Grupo II - cocultura sem citocinas). RESULTADOS: Após sete dias, no grupo I com cocultura confluente, a taxa de proliferação de CNT foi duas vezes maior ao comparar com cocultura subconfluente (35 vs. 16 vezes). No mesmo grupo também foi possível evidenciar elevada taxa de proliferação de células CD133+/CD34+. O índice de proliferação das UFC no grupo I aumentou até oito vezes. A cocultura subconfluente tanto do endotélio vascular do cordão umbilical como do tecido adiposo apresentou menor rendimento em comparação as CTM confluentes. A expansão das células na presença de citocinas apresentou maior proliferação celular ao comparar às coculturas sem adição de citocinas. CONCLUSÃO: Este estudo mostrou que para alto rendimento de células do SCU, o sistema de cocultura requer adição de coquetel de citocinas e CTM confluente independentemente da fonte utilizadaINTRODUCTION: Umbilical cord blood (UCB) hematopoietic stem cells have been successfully used for the treatment of both malignant and non-malignant diseases. Nevertheless, some UCB units could have low total nucleated cells (TNC) dose. Several approaches have been suggested to avoid inadequacy problems of hematopoietic stem cells (HSC) number for transplantation, such as administration of two UCB units to the patient and HSC ex vivo expansion. OBJECTIVE: Evaluate UCB ex vivo expansion proliferative rates in a high and low mesenchymal stem cells (MSC) confluence feeder layer obtained from different MSC sources and by adding or not cytokines cocktail into the medium. METHODS: This study was approved by the Research Ethic Committee (CAPPESQ) of Hospital das Clínicas da Faculdade de Medicina da USP. The collection of UCB (n=10) was made after delivery of the infant and the expulsion of placenta. Processing was performed using volume reduction method which consists in red blood depletion. MSC samples from umbilical cord endothelium were obtained from three different donors and adipose tissue (n=3) obtained from LIM31\'s pattern inventory. The total nucleated cell (TNC), expression of hematopoietic surface markers such as CD133+/CD34+ were observed after seven days of culture. Beyond that, colony forming unit assay (CFU) was performed before and after UCB expansion. The expansion by coculture method was observed in two groups (Group I - coculture with cytokines cocktail added vs. Group II- coculture without cytokines cocktail) for both MSCs sources. RESULTS: After seven days, analysis of confluent coculture showed that TNC proliferation rate ware almost 2 times higher than in subconfluent coculture (35 vs. 16-fold) in Group I and also revealed higher proliferative rate in CD133+/CD34+ cells considering. CFU showed similar increase after seven days of culture in comparison of day 0 (up to 8-fold). Subconfluent coculture for both umbilical cord endothelium and adipose tissue showed lower yield compared with those with high MSC confluence. The expansion in the presence of cytokines showed higher cell proliferation compared to the cocultures without addition of cytokines. CONCLUSION: This study showed that coculture system may require the addition of cytokines cocktail in the media and confluent MSC regardless of source for high yield of UCB cells
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Castro, Mike. „Cytokine Modulation of Cardiomyocyte-Macrophage Interaction“. Wright State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=wright157858331333014.
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Simmers, Phillip Charles. „Benefits of Nitric Oxide Cues to Matrix Synthesis by Healthy and Aneurysmal Human Smooth Muscle Cells within 3D Cocultures“. Cleveland State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=csu1399977973.
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Graffagna, Barry. „Virus Production and Cell Viability of HSV-1-infected Murine Keratinocytes (HEL-30) Co-cultured with Murine Macrophages (RAW 264.7)“. Wright State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=wright1542212790178886.
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Bach, Martin. „Der Einfluss muriner mesenchymaler Stammzellen auf murine zytokin induzierte Killerzellen in der Kokultur“. Doctoral thesis, Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-149957.
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Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required.
In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.
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Ferreira, Luís Pedro Correia Pinto. „Development of multicelular 3D cancer testing platforms for evaluation of new anti-cancer therapies“. Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22713.
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Mestrado em Bioquímica ClínicaO cancro do pulmão (CP) é um dos cancros mais diagnosticados a nível mundial e também um dos mais mortíferos. Atualmente, as terapias administradas a nível clínico para o tratamento do CP são ainda extremamente ineficazes e limitadas no que diz respeito ao aumento da taxa de sobrevivência dos pacientes oncológicos. Esta realidade demonstra a necessidade de investigar ativamente novas terapias para o tratamento desta neoplasia. No entanto a validação pré-clínica de terapias inovadoras para o CP tem-se revelado extremamente difícil devido à inexistência de plataformas que sejam adequadas para testes a nível laboratorial, uma vez que as culturas celulares in vitro bidimensionais (2D), recomendadas pelas agências regulatórias são incapazes de mimetizar as caraterísticas principais dos tumores humanos. Estas limitações têm originado uma fraca correlação entre a performance das terapias nos estudos in vitro e a obtida em ensaios clínicos controlados. Neste contexto, os modelos de tumores tridimensionais (3D) in vitro têm vindo a ser reconhecidos como uma solução para este problema, pois podem recapitular várias componentes do microambiente tumoral. Das várias plataformas 3D in vitro de CP investigadas atualmente muito poucas avaliaram o papel da inclusão de células estaminais mesenquimais (MSCs). Para colmatar esta lacuna, o trabalho de investigação desenvolvido no âmbito desta dissertação descreve a produção e otimização de novos modelos hétero-celulares 3D in vitro. Estas plataformas são compostas por células tumorais do CP (A549) e do seu estroma, nomeadamente fibroblastos da pele e células estaminais mesenquimais derivadas da medula óssea (BM-MSCs). Estes três tipos de células foram co-cultivadas em micropartículas poliméricas de policaprolactona revestidas por ácido hialurónico, com o objetivo de incluir este componente da matriz extracelular que se encontra presente no microambiente do CP. Esta abordagem permitiu formar a nível laboratorial microtecidos multicelulares 3D híbridos que melhor mimetizam a heterogeneidade celular das neoplasias pulmonares. Os resultados obtidos demonstraram que os microtumores formados através da técnica de sobreposição-líquida são reprodutíveis em termos de morfologia e tamanho, apresentaram núcleos necróticos, organização celular 3D e produziram proteínas do microambiente tumoral. Além destas caraterísticas, os dados obtidos através de microscopia de fluorescência revelaram que as BM-MSCs migram para o interior dos microtumores ao longo do tempo. A avaliação da citotoxicidade da Doxorubicina, um fármaco anti-tumoral rotineiramente utilizado a nível clínico, demonstrou que a inclusão de micropartículas aumenta a resistência das células tumorais em modelos homotípicos. Nos modelos tri-cultura heterotípicos a citotoxicidade foi comparável à obtida em microtumores sem micropartículas. Estes resultados evidenciam assim o papel importante dos fibroblastos e das BM-MSCs na resposta dos microtumores. Numa visão global, os modelos 3D formados recapitulam com mais exatidão o microambiente do cancro do pulmão e poderão servir no futuro como plataformas de teste para descobrir ou aperfeiçoar novas terapias, ou combinações de terapêuticas, para este tipo de neoplasia.
Lung cancer (LC) is one the most commonly diagnosed cancers worldwide, being also one of the deadliest. Currently, clinically administered therapies for treatment of LC are still extremely ineffective and limited in increasing oncologic patients survival rates. This reality evidences the necessity of actively investigating novel therapies for the treatment of LC. However, preclinical validation of novel therapies as revealed itself as an extremely arduous process, due to the lack of suitable laboratory testing platforms since the recommend in vitro bi-dimensional (2D) cell cultures are unable to fully mimic the main hallmarks of human tumors. In this context, in vitro tridimensional (3D) tumor models are being increasingly recognized as a solution due to their ability to correctly recapitulate several characteristics of the tumor microenvironment (TME). Amongst currently developed 3D in vitro platforms for the study of LC, few have included or studied the role of mesenchymal stem cells (MSCs). To provide further insights into this hypothesis, the research work developed in this thesis describes the production and optimization of novel heterotypic in vitro 3D models, comprised by non-small-cell lung cancer cells (A549) and stromal cells, namely skin fibroblasts (HFs), and bone-marrow derived mesenchymal stem cells (BM-MSCs). These three diverse cell populations were co-cultured in hyaluronic acid coated polymeric polycaprolactone microparticles (LbL-MPs) as to include this key extracellular matrix component of LC TME. This approach allowed the formation of 3D multicellular heterotypic microtissues (3D-MCTS) that better recapitulate the cellular heterogeneity of LC TME in the laboratory. The obtained findings demonstrate that these models formed via the liquid-overlay technique were reproducible in terms of morphology and size, presented necrotic core formation, 3D cellular organization, and deposited matrix proteins in a similar manner as in the TME. Besides this, fluorescence microscopy data revealed that BM-MSCs migrated overtime into the microtumors core . Performed doxorubicin in vitro cytotoxicity assays revealed that the inclusion of LbL-MPs lead to an increased resistance of homotypic A549 monoculture models against this anti-cancer drug commonly used in clinical treatments. Alongside, the cytotoxicity obtained in triculture heterotypic models was comparable to that of microtumors without LbL-MPs inclusion, showcasing the role of HFs and BM-MSCs in microtumors response to therapy. Globally, the herein bioengineered 3D models were able to recapitulate with an increased precision the TME of LC, making them suitable test platforms for development or improvement of standalone or combinatorial therapies for this type of neoplasia.
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Jing, Duohui. „Mobilisation, Isolation and Coculture of Haematopoietic Stem Cells“. Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-39915.
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Since decades, hematopoietic stem cell transplantation (HSCT) has become a well established treatment modality for hematological malignancies and non-malignant disorders. Autologous and allogeneic hematopoietic stem cells (HSCs) mobilized into the peripheral blood (PB) have been used as a preferred source of transplantable stem cells1-3. And umbilical cord blood (UCB) has been introduced as a more attractive HSC source for HSCT, because fetal stem cells in UCB are speculated to be more primitive in comparison to adult stem cells. However the limited amount of HSCs is limiting their application for stem cell therapy in clinic. Therefore, people started to utilize extra-embryonic tissue to harvest more fetal stem cells, while people also tried to optimize the clinical protocol to mobilize more adult stem cells out of adult bone marrow. The innovative strategies and feasible procedures were discussed in this thesis.
The axis of the chemokine receptor CXCR4 and its ligand SDF-1 is important for trafficking and homing of HSCs. It has already been demonstrated that the bicyclam AMD3100, a CXCR4 antagonist, in combination with G-CSF is able to induce a significant mobilization of CD34+ cells4. And human placenta is a potent hematopoietic niche containing hematopoietic stem and progenitor cells throughout development5. The homing of HSCs to the placenta is probably also mediated by the expression of SDF-1 as demonstrated for the bone marrow niche. In this study (part 1 of the chapter “Results and discussions”), we utilized AMD3100 to mobilize HSCs from placenta. And we can demonstrate that the CXCR4 antagonist AMD3100 mobilise placenta derived CD34+ cells ex utero already after 30 min of incubation and may further enhance the efficacy of harvesting placenta-derived HSC.
The alpha4 integrin CD49d is involved in migration and homing of hematopoietic stem cells (HSC). Therapeutic application of natalizumab, an anti-CD49d antibody, in patients with multiple sclerosis (MS) has been associated with increased levels of circulating CD34+ progenitors. In our study (part 2 of the chapter “Results and discussions”), we compared circulating HSCs from MS patients after natalizumab treatment and HSCs mobilized by G-CSF in healthy volunteers, with regard to their migratory potential, clonogenicity and gene expression. CD34+ cells in the blood and marrow of natalizumab-treated patients expressed less of the stem cell marker CD133, were enriched for erythroid progenitors (CFU-E) and expressed lower levels of adhesion molecules. The level of surface CXCR-4 expression on CD34+ cells from patients treated with natalizumab was higher compared to that of CD34+ cells mobilized by granulocyte-colony stimulating factor (G-CSF) (median 43.9% vs. 15.1%). This was associated with a more than doubled migration capacity towards a chemokine stimulus. Furthermore, CD34+ cells mobilized by natalizumab contained more m-RNA for p21 and less MMP9 compared to G-CSF mobilised HSC. Our data indicate that G-CSF and CD49d blockade mobilize different HSC subsets and suggest that both strategies may be differentially applied in specific cell therapy approaches.
In order to further improve the clinical outcome of HSC transplantation, many groups are focusing on ex vivo maintain or expand HSC. Unfortunately, the maintenance of HSC in vitro is difficult to achieve because of their differentiation. This is presumably caused by a lack of appropriate cues that are provided in vivo by the microenvironment. Indeed, HSCs located in the bone marrow are interacting with a specific microenvironment referred to as the stem cell niche, which regulates their fate in terms of quiescence, self-renewal and differentiation. An orchestra of signals mediated by soluble factors and/or cell-to-cell contact keeps the balance and homeostasis of self-renewal, proliferation and differentiation in vivo. To investigate the communication between HSCs and the niche, coculture assays with mesenchymal stromal cells (MSCs) were performed in vitro. Here, we can demonstrate that cell-to-cell contact has a significant impact on hematopoietic stem cells expansion, migratory potential and stemness. In this study (part 3 of the chapter “Results and discussions”), we investigated in more detail the spatial relationship between hematopoietic stem cells and mesenchymal stromal cells during ex-vivo expansion. And we defined three distinct localizations of HSCs relative to MSC layer: (i) those in supernatant (non-adherent cells); (ii) cells adhering on the surface of mesenchymal stromal cells (phase-bright cells) and (iii) cells beneath the mesenchymal stromal cells (phase-dim cells). Our data suggest that the mesenchymal stromal cell surface is the dominant location where hematopoietic stem cells proliferate, whereas the compartment beneath the mesenchymal stromal cell layer seems to be mimicking the stem cell niche for more immature cells. Our data provide novel insight into the construction and function of three-dimensional HSC–MSC microenvironments.
In summary, we provided a new method to isolate fetal stem cells from extra-embryonic tissue (i.e. placenta) in the first part, then we discussed an innovative strategy with CD49d blockade to improve clinical modality for adult stem cell mobilization in the second part, and finally we investigated HSC maintenance and expansion in vitro and provided feasible way to mimic HSC niche in vitro in the last part.
This thesis contributes to HSC-based stem cell therapy in two aspects, i.e. 1) fetal and adult stem cell isolation holding great therapeutic potential for blood diseases; 2) ex vivo stem cell manipulation providing a valuable platform to model HSC niche regulation.
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Souidi, Naima. „Protektion humaner endothelialer Vorläuferzellen durch die Koapplikation mit Mesenchymalen Stamm-/Vorläuferzellen“. Doctoral thesis, Humboldt-Universität zu Berlin, 2017. http://dx.doi.org/10.18452/18608.
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Endothelzell-basierte Therapien vermitteln regenerative Effekte hinsichtlich der Revaskularisierung von ischämischen Geweben. Doch ist die Verfügbarkeit von autologen Endothelzellen aufgrund einer krankheitsbedingt reduzierten Frequenz im peripheren Blut oder einer verminderten Integrität der endogenen Endothelzell-Populationen eingeschränkt. Hingegen ist es möglich, allogene endotheliale Vorläuferzellen aus der Nabelschnur in zelltherapeutisch relevanten Mengen zu isolieren.
In der vorliegenden Arbeit wurden zunächst die Eigenschaften allogener humaner Nabelschnur (NS)-abgeleiteter sog. Endothelial Colony-Forming Cells (ECFCs) mit denen von venösen NS-abgeleiteten Endothelzellen verglichen. Aufgrund der nachgewiesenen Immunogenität von allogenen ECFCs wurde eine weiterführende Strategie zur Reduktion dieser immunogenen Eigenschaften durch die Koapplikation mit Mesenchymalen Vorläuferzellen (MSCs) verfolgt.
Humane ECFCs wurden mit MSCs desselben Spenders kombiniert und in funktionellen in vitro- und in vivo-Assays untersucht. Dadurch konnte nachgewiesen werden, dass IFNγ-stimulierte ECFC/MSC-Kokulturen eine reduzierte Expression von HLA-Molekülen zeigen. Entsprechend induzierten spezifische CD8+ T-Zellen eine reduzierte Lyse der kokultivierten ECFCs und MSCs. Die Kokultur von ECFCs und MSCs mit allogenen Immunzellen führte zu einer nahezu vollständigen Inhibition der T-Zell-Proliferation. Um die reduzierte Immunogenität von ECFC und MSC in vivo zu verifizieren, wurden die Zellen in immundefiziente Mäuse injiziert, welche nachfolgend mit humanen PBMCs rekonstituiert wurden. So konnte nachgewiesen werden, dass die Koapplikation von ECFCs und MSCs nicht nur die Entstehung von stabilen Gefäßnetzwerken begünstigt, sondern zudem in den Transplantaten zu einer verringerten Immunzell-Infiltration führte. Die Koapplikation von ECFCs mit MSCs könnte daher eine klinische Nutzung dieser allogenen Quelle für die therapeutische Unterstützung der Vaskularisierung ermöglichen.Endothelial cell-based therapies promote tissue regeneration and vascularization after ischemic damage. The availability of autologous endothelial progenitor cells is restricted in diseased patients, however therapeutically relevant numbers of allogeneic Endothelial Progenitor Cells can be isolated from an umbilical cord (UC). In the present study, the immunogenic properties of these Endothelial Colony Forming Cells (ECFCs) were first compared to human umbilical vein endothelial cells (HUVECs). Both cytokine-treated endothelial cells induced CD4+ and CD8+ T cell proliferation after coculture with allogeneic immune cells. So far, the potential interactions between ECFCs and Mesenchymal Stem/Progenitor Cells (MSCs) concerning their immunological features is poorly understood, but we hypothesize that MSCs might improve the immune compatibility and vessel building characteristics of ECFCs. Therefore, human UC-derived ECFC and MSC cocultures from the same donor were analyzed using various functional in vitro and in vivo assays. Stimulation of these cocultures with IFNγ caused strongly reduced expression levels of HLA-molecules compared to ECFC monocultures. The decreased molecular density on the cocultured ECFCs resulted in reduced cytotoxic CD8+ T cell-mediated lysis. Further, during IFNγ stimulation, the combination of ECFCs with MSCs prevented initiation of allogeneic T cell proliferation. To verify this concept in vivo, ECFCs and MSCs were co-transplanted in a humanized allograft mouse model in immunodeficient mice in order to effectively induce stable microvessels. These experiments demonstrate that when MSCs are co-applied with ECFCs, they not only support the formation of stable blood vessels, but also lead to fewer HLA-DR+ human vascular structures and fewer infiltrating human leukocytes. The data presented indicate that crosstalk between UC-derived ECFCs and MSCs might lower the risk of allogeneic ECFC rejection.
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Hespeling, Ursula, Kurt Jungermann und Gerhard P. Püschel. „Feedback-inhibition of glucagon-stimulated glycogenolysis in hepatocyte/kupffer cell cocultures by glucagon-elicited prostaglandin production in kupffer cells“. Universität Potsdam, 1995. http://opus.kobv.de/ubp/volltexte/2008/1669/.
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Prostaglandins, released from Kupffer cells, have been shown to mediate the increase in hepatic glycogenolysis by various stimuli such as zymosan, endotoxin, immune complexes, and anaphylotoxin C3a involving prostaglandin (PG) receptors coupled to phospholipase C via a G(0) protein. PGs also decreased glucagon-stimulated glycogenolysis in hepatocytes by a different signal chain involving PGE(2) receptors coupled to adenylate cyclase via a G(i) protein (EP(3) receptors). The source of the prostaglandins for this latter glucagon-antagonistic action is so far unknown. This study provides evidence that Kupffer cells may be one source: in Kupffer cells, maintained in primary culture for 72 hours, glucagon (0.1 to 10 nmol/ L) increased PGE(2), PGF(2 alpha), and PGD(2) synthesis rapidly and transiently. Maximal prostaglandin concentrations were reached after 5 minutes. Glucagon (1 nmol/L) elevated the cyclic adenosine monophosphate (cAMP) and inositol triphosphate (InsP(3)) levels in Kupffer cells about fivefold and twofold, respectively. The increase in glyco gen phosphorylase activity elicited by 1 nmol/L glucagon was about twice as large in monocultures of hepatocytes than in cocultures of hepatocytes and Kupffer cells with the same hepatocyte density. Treatment of cocultures with 500 mu mol/L acetylsalicylic acid (ASA) to irreversibly inhibit cyclooxygenase (PGH-synthase) 30 minutes before addition of glucagon abolished this difference. These data support the hypothesis that PGs produced by Kupffer cells in response to glucagon might participate in a feedback loop inhibiting glucagon-stimulated glycogenolysis in hepatocytes.
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Huppert, Cécile. „Développement d’un modèle de coculture cellules dendritiques lymphocytes T pour l’évaluation du danger des substances sensibilisantes“. Thesis, Université de Lorraine, 2018. http://www.theses.fr/2018LORR0195/document.
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Les allergies représentent un problème majeur dans le domaine des maladies professionnelles et ont un impact sérieux sur la vie des travailleurs. Les allergies professionnelles sont principalement cutanées et respiratoires ; elles peuvent être causées par des produits chimiques de bas poids moléculaire. Dans le passé, les tests destinés à identifier les produits susceptibles d’entraîner des allergies étaient réalisés sur l’animal. Or, la législation européenne engage à limiter le recours à l’expérimentation animale pour évaluer le pouvoir sensibilisant des substances chimiques, incitant à développer des tests in vitro de substitution. C’est dans ce contexte que nous avons cherché à développer des modèles de cultures cellulaires destinés à identifier les substances sensibilisantes. Un premier modèle utilisant des cellules dendritiques dérivées de moelle osseuse (BMDC) de souris BALB/c a été développé et a donné des résultats prometteurs pour l’identification des produits sensibilisants et leur catégorisation selon leur puissance sensibilisante. De plus, la voie de signalisation Nrf2/Keap1 semble être impliquée dans la réponse de ce modèle cellulaire aux sensibilisants. Dans le but de compléter ce modèle et d’évaluer la capacité des BMDC à activer les lymphocytes T (LT), un modèle de coculture de BMDC et LT a été mis au point avec un sensibilisant de référence avant d’être testé sur un ensemble de produits de référence (sensibilisants cutanés et respiratoires, irritants et non sensibilisants). Les BMDC de notre modèle, exposées à des sensibilisants, se sont révélées capables d’activer les LT en coculture. Enfin, des essais préliminaires utilisant des cellules de souris de souche C57BL6/J dans notre modèle de coculture ont donné des résultats comparables à ceux obtenus avec des cellules issues de la souche BALB/c. Les modèles de cultures cellulaires BMDC et de coculture BMDC-LT sont prometteurs dans le cadre du développement de méthodes de substitution à l’expérimentation animale pour l’évaluation du pouvoir sensibilisant de substances chimiquesAllergies constitute an important issue in the field of occupational health and have a serious impact on the lives of workers. Occupational allergies are mainly contact and respiratory allergies and can be caused by low molecular weight chemicals. In the past, the tests that were used to identify the potential allergens were carried out on animals. However, European legislation has provided the impetus for reducing the use of animal testing to assess the sensitization potential of chemicals and promoted the development of alternative in vitro tests. In this context, we aimed to develop cell culture models to identify sensitizers. A first model using bone marrow derived dendritic cells (BMDC) from BALB/c mice was developed and showed promising results for identifying sensitizers and classify them according to their allergenic potency. Moreover, the Nrf2/Keap1 pathway seems to be involved in the response of this cell model to sensitizers. In order to supplement this model and to assess the functionality of BMDC, a BMDC-T cell (TC) coculture model was developed with a reference sensitizer before being tested on a range of reference sensitizers (cutaneous and respiratory sensitizers, irritants and non-sensitizers). The BMDC of our model, while exposed to sensitizers, were able to activate TC in coculture. Finally, preliminary tests using the cells of C57BL6/J mice in our coculture model showed that similar results to those obtained with cells from the BALB/c strain. The models of BMDC cultures and BMDC-TC coculture are promising for the development of alternative methods to animal experimentation assessing the sensitizing potential of chemicals
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Danoy, Mathieu. „Development of a physiologically-relevant in-vitro microfluidic model for monitoring of pancreatic cancer cells interactions with the liver“. Thesis, Lille 1, 2017. http://www.theses.fr/2017LIL10093/document.
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Le procédé de la métastase cancéreuse et sa compréhension sont devenus un des sujets majeurs de recherche en Biologie. En utilisant des modèles in-vitro en culture statique et dynamiques, nous avons étudié la possibilité de reproduire l’environnement physiologique in-vivo avec ces modèles. Nous avons développé un modèle de coculture hiérarchique dans des plaques à fond en PDMS. Composé d’hépatocytes, de pericytes et de cellules endothéliales. Dans différentes conditions, l’influence de ces cellules sur l’adhésion de cellules cancéreuses ou promyéloblastiques a été analysée ainsi que leur effet sur l’état inflammatoire du système. Afin de reproduire le flux sanguin et les forces de cisaillement présents in-vivo, le modèle a été transféré dans un système microfluidique. Le système se compose de trois canaux séparés par des micro-piliers, pouvant être remplis indépendamment. Les pericytes insérés dans du gel, les hépatocytes, les cellules endothéliales et finalement les cellules cancéreuses ont été injectés de façon successive afin de reproduire l’environnement in-vivo. Les cellules ont été trouvées viables durant toute la culture et des marqueurs relatifs au foie et à l’inflammation exprimés. L’influence des hépatocytes et des pericytes a été analysé. Il a été observé que les cellules cancéreuses adhérées dans le canal du haut étaient attirées par les autres cellules dans les diffèrent canaux. Les modèles établis posent de solides bases pour d’autres systèmes plus complexes et d’intérêt pouvant servir de complément aux modèles in-vivo lors de la recherche de nouvelles substances médicamenteusesThe cancer metastatic process and its understanding have been a major topic of interest for researchers in the past. Using in-vitro models in both standard culture conditions and in microfluidic devices, we investigated the feasibility of such models in the representation of the physiological in-vivo situation. We developed a hierarchical coculture model in PDMS plates, composed of hepatocytes, pericytes and endothelial cells. In different culture conditions, the influence of the different cells composing the model on the adhesion of cancer cells and promyeloblastic cells was investigated as well as the influence on the inflammatory state of the culture. To reproduce the in-vivo blood flow and shear stress to which the endothelial cells and the adhering cells are subjected, the model was then transferred into a microfluidic biochip. The device was composed of three channels, separated by micropillars and which could be filled independently one from another. Pericytes embedded in a hydrogel, hepatocytes, endothelial cells and finally pancreatic cancer cells could be inserted successively to reproduce the in-vivo hierarchical situation. Cells were found to viable after the culture and markers related to the liver and inflammation to be expressed. The influence of the presence of hepatocytes and pericytes was investigated by varying the culture conditions. It was found that pancreatic cancer cells were attracted by the cells in other channels in coculture. The established models lay the bases for more complex and relevant systems that could complement their in-vivo counterparts in the drug discovery process
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Baudequin, Timothée. „Caractérisation biologique et mécanique d'un subsitut osseux biohybride et développement de scaffolds par électrospinning : vers un pansement vivant pour la reconstruction maxillo-faciale“. Thesis, Compiègne, 2015. http://www.theses.fr/2015COMP2219/document.
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Un substitut osseux hybride, composé d’un biomatériau support (scaffold) et de cellules vivantes, a été étudié, développé par la méthode d’ingénierie tissulaire et caractérisé. Il devait répondre aux attentes spécifiques de la chirurgie maxillofaciale : un protocole standard pouvant s’adapter aux géométries complexes des défauts osseux de chaque patient, une forme souple et manipulable, une pré-vascularisation et une cohésion mécanique suffisante. Une forme de feuillet fin et plat a ainsi été définie et développée au sein d’une chambre de culture parallélépipédique spécifique, en utilisant une monocouche de granules de phosphate de calcium comme support. Après une caractérisation biologique et mécanique complète à partir d’une lignée cellulaire, le procédé a été validé puis transposé à une coculture de cellules primaires humaines (cellules souches et endothéliales). La bonne différenciation et la pré-vascularisation ont été constatées mais le maintien mécanique pouvait être considéré comme insuffisant pour assurer une manipulation en cours d’opération chirurgicale. La dernière partie de ce travail de thèse a donc consisté dans la mise en place d’un montage de production de fibres électrospinnées et leur utilisation comme nouveau support de culture. La formation de ces matériaux a été rendue opérationnelle de façon optimale pour différents polymères. Leur potentiel en tant que scaffold favorisant la différenciation en os ou en tendon a été vérifié et comparé à d’autres matériaux fibreux obtenus dans le cadre de collaborations nationales et internationales. La faisabilité de l’application de sollicitations mécaniques aux substituts en cours de culture a également été étudiéeAn hybrid bone substitute, based on a specific biomaterial (scaffold) and living cells, was studied, developed with a tissue engineered method and characterized. It should meet the expectations of the maxillofacial surgery : a standard process which could fit with the complex geometries of each patient’s bone mass loss, a flexible shape with an easy handling, a prevascularization and a sufficient mechanical cohesion. A sheet-like shape was thus designed and developed in a specific flat cell culture chamber, with a monolayer of calcium phosphate granules as a scaffold. After both biological and mechanical full characterizations with a cell line, the process was adapted to a coculture of human primary cells (stem and endothelial cells). Relevant differentiation and prevascularization were highlighted but the mechanical cohesion could be noticed as too low to ensure an easy handling during the surgery. The last part of this thesis project was thus the set-up of a device for electrospun polymer fibers in order to use them as a new scaffold. The production of these materials was efficiently performed for several polymers. The differentiation potential for bone and tendon lineages was studied and compared to other scaffolds from national and international collaborations. The application of mechanical solicitations to the substitutes during cellculture was also studied
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Kunigal, Sateesh Sreenivasan. „Urokinase-activated Stat1 mediates antiproliferative effect in vascular smooth muscle cells cocultered with monocytes“. [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=971935858.
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SUFI, BIANCA da S. „Utilização de cocultura de melanócitos e queratinócitos para avaliação da ação do líquido da castanha de caju (LCC) na pigmentação epidérmica“. reponame:Repositório Institucional do IPEN, 2013. http://repositorio.ipen.br:8080/xmlui/handle/123456789/10197.
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Dissertação (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
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Moritz, Joseph M. (Joseph Michael). „Increased differentiation properties in two- and three-dimensional coculture of hepatocytes and liver epithelial cells by a novel quantitative functional liver assay“. Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/39352.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2007.Includes bibliographical references (leaves 100-121).
Hepatic stem cells in adult rats are activated by chemical injury to the liver, causing hepatic progenitor cells to proliferate, integrate into the hepatic plates, and differentiate into hepatocytes. In an attempt to model this process in vitro, we established and quantitatively assayed the differentiation properties of a strain of rat liver epithelial cells (LEC), lig8, grown in coculture with mature liver cells in a three dimensional, perfused microreactor optimized for hepatocyte culture. Lig8 was derived by suppression of the asymmetric growth kinetics that may be indicative of stem cells, and Lig8 progeny can be induced to exhibit several hepatocyte-specific differentiation properties in vitro; however, Lig8 full hepatocyte functional differentiation in culture has not yet been achieved. We hypothesized that more extensive differentiation properties may be observed in vitro if the Lig8 cells are cultured in an engineered analog of the 3D tissue environment that influences progenitor cell differentiation in vivo. We also assayed the differentiation properties of the hepatocytes in coculture. Previous studies have shown an increase in the differentiation of hepatocytes in 2D hepatocyte-LEC cocultures; we wished to determine if the benefit of coculture also occurs in the 3D microreactor.
(cont.) We therefore compared the differentiation properties of both cell types in 3D microreactor cocultures to three more traditional culture formats: 2D rigid collagen monolayer, 2D collagen gel sandwich, and 3D spheroids. To assess the functional differentiation state of both cell types in these cocultures, we implemented a cell-localizable quantitative assay for endocytotic uptake of fluorescent ligands of the hepatocyte asialoglycoprotein receptor (ASGPR). T'o additionally assay overall differentiation of the cultures, we examined the level of expression compared to in vivo of three hepatocyte-specific transcripts: ASGPR, and two highly abundant drug-metabolic enzymes CYP3A1 and CYP2E1. Of all the culture modes tested, three-dimensional microreactor coculture was shown to be the most highly differentiated by the fluorescent ligand uptake assay for ASGPR and CYP3A1, with near in vivo expression of CYP3A1. However, coculture only improved the expression of the transcripts for ASGPR and CYP2E1 in 2D rigid collagen monolayer cocultures. lig8 exhibited no uptake of the ASGPR-ligand in monoculture, but in all cocultures tested, rare cells were found positive, with a higher percentage of lig8 taking up the ligand in 31) than in 2D (although cell fusion was not ruled out).
(cont.) We conclude that this three-dimensional coculture system may be more physiological in vitro model for the study of LEC-mature cell interactions and liver response to carcinogens.
by Joseph M. Moritz.
Ph.D.
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Ziegler, Elke [Verfasser], Carsten [Akademischer Betreuer] Gründker und Matthias [Akademischer Betreuer] Dobbelstein. „Characterization of human breast cancer cells affected by coculture conditions and kisspeptin-10 / Elke Ziegler. Gutachter: Carsten Gründker ; Matthias Dobbelstein. Betreuer: Carsten Gründker“. Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2013. http://d-nb.info/1044414847/34.
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Carra, Bruna Beatriz Gimenez. „Avaliação da polarização de macrófagos em coculturas com células de Schwann infectadas pelo Mycobacterium leprae“. Botucatu, 2018. http://hdl.handle.net/11449/164790.
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Orientador: Vânia Niéto Brito de SouzaResumo: A infecção pelo Mycobacterium leprae (ML) estimula um processo de desdiferenciação e proliferação das células de Schwann (SCs) que pode contribuir para a disseminação do bacilo. Os macrófagos (MOs) são células efetoras da resposta imune que promovem a eliminação de patógenos, entretanto, na hanseníase são colonizados pelo ML. Sabe-se que os MOs podem apresentar uma polarização funcional na qual os MOs M1 apresentam características pró-inflamatórias e microbicidas enquanto os MOs M2 atuam na reparação tecidual e possuem perfil anti-inflamatório. SCs infectadas pelo ML produzem mediadores capazes de interferir com a função dos MOs aumentando sua sobrevida e promovendo sua migração. Embora diferentes programas funcionais tenham sido observados em MOs de pacientes com formas polares da hanseníase a influência de SCs nesse processo não é sabida. Neste estudo avaliamos se SCs infectadas pelo ML podem interferir na polarização de MOs murinos derivados de medula óssea. Para tanto, culturas primárias de SCs murinas foram infectadas experimentalmente com bacilos viáveis e cocultivadas com MOs. Nossos resultados indicam que a produção de óxido nítrico foi baixa nas culturas de MOs após a infecção com o bacilo, mas mostrou-se aumentada nas coculturas de MOs e SCs infectadas pelo ML. A infecção com ML não induziu produção significante das citocinas IL-6, IL-10 e TNF em culturas de MOs e SCs, entretanto, a interação entre MOs e SCs infectadas com o bacilo resultou em aumento na produção de... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Mycobacterium leprae (ML) infection stimulates dedifferentiation and proliferation of Schwann cells (SCs) that may contribute to the spread of the bacillus. Macrophages (MOs) are effector cells of the immune response that promote the elimination of pathogens, however, in leprosy they are colonized by ML. It is known that MOs can present a functional polarization in which M1 MOs show pro-inflammatory and microbicidal activities while M2 MOs act in tissue repair presenting an anti-inflammatory profile. SCs infected by ML produce mediators able to interfere with MOs function, increasing their survival and promoting their migration. Although different functional programs have been observed in MOs from patients with polar forms of leprosy, the influence of SCs in this process is not known. In this study we evaluated whether SCs infected with ML could interfere in the polarization of murine MOs derived from bone marrow. For this purpose, primary cultures of murine SCs were experimentally infected with viable bacilli and co-cultivated with MOs. Our results indicate that nitric oxide production was low in cultures of MOs after infection with the bacillus, but it was increased in the co-cultures of MOs and ML-infected SCs. The infection with ML did not induce significant production of IL-10, TNF and IL-6 in cultures of MOs and SCs, however, the interaction between MOs and ML infected-SCs resulted in increased production of cytokines, mainly IL-10, inducing a decrease in the TNF/IL-10 ... (Complete abstract click electronic access below)
Mestre
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Hittinger, Marius [Verfasser], und Claus-Michael [Akademischer Betreuer] Lehr. „Autologous coculture of primary human alveolar epithelial cells and macrophages for evaluating the safety and efficacy of novel inhalation pharmaceuticals / Marius Hittinger ; Betreuer: Claus-Michael Lehr“. Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2017. http://d-nb.info/1127040006/34.
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Chebbo, Mohamad. „Rôle des plaquettes dans l’asthme sévère“. Thesis, Aix-Marseille, 2022. http://www.theses.fr/2022AIXM0229.
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Les plaquettes sont des cellules anucléées qui jouent un rôle dans l'hémostase, la thrombose et l’immunité. Elles sont également impliquées dans la pathogenèse de certaines maladies inflammatoires telles que l'asthme. Le but de ce travail est de mieux comprendre le rôle des plaquettes dans l’asthme sévère en étudiant : 1) le transcriptome des plaquettes circulantes de patients asthmatiques sévères et en évaluant l’expression plaquettaire, chez ces patients, d’HLA-F, une molécule mobilisée à la surface de différents types cellulaires après leur activation 2) la présence des plaquettes dans les bronches, et en modélisant l’interaction entre les plaquettes et les cellules épithéliales bronchiques par une coculture in vitro. Dans la première partie, j’ai tout d’abord mis au point une technique de purification des plaquettes montrant la nécessité d’éliminer les leucocytes et les érythrocytes résiduels qui altèrent considérablement les résultats du transcriptome plaquettaire. Ensuite, j’ai comparé le transcriptome des plaquettes des patients asthmatiques sévères avec celui des plaquettes de sujets contrôles. J’ai ainsi pu montrer que le transcriptome plaquettaire des patients asthmatiques sévères est différent de celui des témoins. Parallèlement, j’ai montré qu’HLA-F était plus fortement exprimé à la surface des plaquettes des patients asthmatiques sévères à l’état de base et qu’HLA-F pouvait être mobilisé à leur surface suite à une activation. Dans la deuxième partie, j’ai pu montrer que les plaquettes sont présentes dans la sous-muqueuse bronchique et que les plaquettes activées induisent la sécrétion du facteur de croissance PDGF-BB par les cellules épithéliales bronchiquesPlatelets are anucleate cells that play a role in hemostasis, thrombosis and immunity. They are also involved in the pathogenesis of some inflammatory disorders such as asthma. The aim of this work is to better understand the role of platelets in severe asthma by studying: 1) the transcriptome of circulating platelets from severe asthma patients and by evaluating the platelet expression, in these patients, of HLA-F, a molecule mobilized to the surface of different cell types after their activation. 2) the presence of platelets in the bronchi, and by modeling the interaction between platelets and bronchial epithelial cells in an in vitro coculture system. In the first part, I developed a platelet purification technique showing the need to remove residual leukocytes and erythrocytes that significantly alter the platelet transcriptome results. Then, I compared the platelet transcriptome from severe asthmatic patients with that of platelets from control subjects. I was able to show that the platelet transcriptome of severe asthmatic patients is different from that of controls. Simultaneously, I showed that HLA-F was more strongly expressed on the surface of platelets from severe asthma patients at steady state and that HLA-F could be mobilized to their surface following activation. In the second part, I was able to show the presence of platelets in the bronchial submucosa. In addition, the co-culture between bronchial epithelial cells and platelets allowed to demonstrate a secretion of the growth factor PDGF-BB by bronchial epithelial cells, specifically induced by activated platelets
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Honda, Suzana Terumi. „Influência de fibroblastos de linfonodo axilar de pacientes com câncer de mama na expressão gênica de células mamárias malignas MDA-MB231 e MCF-7“. Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-04042016-095339/.
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O comportamento do câncer de mama pode ser influenciado pela interação entre células tumorais e estromais que infiltram e circundam o tumor. Acredita-se também que as células que compõem o microambiente linfonodal possam influenciar o perfil da expressão gênica de células mamárias malignas MDA-MB231, induzindo ou inibindo o desenvolvimento de metástase regional. Para avaliar essa possibilidade, células MDA-MB231 foram co-cultivadas com fibroblastos obtidos de linfonodos comprometidos ou não comprometidos de pacientes com câncer de mama, separadas por membranas porosas por 72 horas. O perfil da expressão gênica foi avaliado através de uma plataforma de cDNA microarray de 4608 genes. Observamos que 155 e 188 genes foram modulados em células MDA-MB231 na presença de fibroblastos de linfonodos não comprometidos e comprometidos, respectivamente, quando comparados com células MDA-MB231 cultivadas isoladamente. Análise do agrupamento hierárquico não supervisionado utilizando os genes diferencialmente expressos revelou a presença de dois grupos, um constituído por células MDA-MB 231 em monocultura e outro por células mantidas em co-cultura com fibroblastos. Splicing de RNAm, via spliceossoma, ciclo celular e regulação da transcrição por promotores da RNA polimerase II, foram algumas funções moduladas em células MDA-MB231. A seguir novos ensaios de co-cultura foram realizados e a expressão de alguns genes foram analisados por RT-PCR. FN3K e COMT foram menos expressos em células MDA-MB231 na presença de fibroblastos de linfonodos, mas não em co-cultura com células MCF-7, nas quais apenas HTRA1 mostrou-se hipoexpresso em casos de co-cultura com fibroblastos. Esses resultados sugerem que a inter-relação entre células estromais e células tumorais são dependentes do subtipo do tumor, de fenótipo luminal ou basalBreast cancer behavior may be influenced by interactions between cancer and stromal cells, which infiltrate and surround the tumor. It is also believed that cells within the lymph node microenvironment may influence the gene expression profile of breast cancer cells, stimulating or inhibiting regional metastasis development. To evaluate this possibility, breast cancer MDAMB231 cells were co-cultured with fibroblasts obtained from involved or uninvolved lymph nodes from breast cancer patients, using a porous membrane, for 72 hours. Gene expression profile was evaluated through a cDNA microarray platform containing 4608 genes. MDA-MB231 cells had 155 and 188 genes modulated by the presence of fibroblasts from uninvolved or involved nodes, respectively, as compared to MDA-MB231 cells cultured alone. Unsupervised hierarchical clustering using the differentially expressed genes revealed the presence of two groups, one comprising MDA-MB231 cells cultured aloned and another one, MDA-MB231 cells co-cultured with fibroblasts. Nuclear mRNA splicing, via spliceosome, cell cycle, and regulation of transcription from RNA polymerase II promoter, were functions regulated in cocultured MDA-MB231 cells. New co-culture assays were performed and expression of a few genes was further evaluated by RT-PCR. FN3K and COMT were both down-regulated in MDA-MB231 cells in the presence of nodes\' fibroblasts, but not in co-cultured MCF7 cells, while HTRA1 was just down regulated in MCF7 co-cultured cells. These results suggest that the interrelationship between stromal and cancer cells are dependent on the subtype of tumor, whether from luminal or basal-like phenotype
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Qin, Hong. „Co-culture of hepatocytes with mesenchymal stem cells for cellular therapy in liver disease“. Thesis, King's College London (University of London), 2014. https://kclpure.kcl.ac.uk/portal/en/theses/coculture-of-hepatocytes-with-mesenchymal-stem-cells-for-cellular-therapy-in-liver-disease(7d1b05f4-343c-4f98-93fa-ae23a86788c2).html.
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A major hurdle facing current hepatocyte transplantation practice is the marginal quality of isolated hepatocytes. Previous studies showed that mesenchymal stem cells (MSCs) could maintain morphology and improve liver-specific metabolism of co-cultured hepatocytes. The present work aimed to optimise the MSCs co-culture system by testing adipose tissue (AT), bone marrow, and umbilical cord-derived MSCs at predefined seeding ratios. Liver-specific metabolism and apoptosis assays were performed to investigate hepatotrophic and antiapoptotic effects of MSCs co-culture. Indirect co-culture was established to investigate the role of paracrine factors in hepatotrophic effect of MSCs co-culture. Hypoxia-preconditioned (HPc) MSCs were co-cultured with hepatocytes to investigate potentiative effect of HPc induction. Intracellular reactive oxygen species (ROS) activity quantitation and antagonisation experiments were performed to investigate whether HPc potentiated MSCs co-culture by an intracellular ROS-dependent mechanism. Tumour necrosis factor alpha (TNF-α), transforming growth factor beta1 (TGF-β1), extracellular collagen, and apoptosis- associated caspase and BAX/BCL-2 signalling pathways were analysed to investigate the contribution of soluble factors, extracellular collagen, and gene signalling to the hepatotrophic effects of MSCs co-culture and potentiative effect of HPc induction. All the three types of MSCs exhibited a similar hepatotrophic effect, with a comparable effect even in low-density AT-MSCs co- culture. Hepatotrophic and antiapoptotic effects of MSCs showed a cell contact dependent manner, and HPc potentiated MSCs co-culture by a cell-contact intracellular ROS-dependent mechanism. Decreased hepatocyte autocrine TNF-α, increased MSC autocrine TGF-β1, and enhanced MSCs deposition of extracellular collagen contributed to the hepatotrophic effects of MSCs co-culture and potentiative effect of HPc induction, with downregulated expression of proapoptotic CASP9, BAX, and BID and upregulated expression of antiapoptotic BCL-2. It is concluded that synergistic effects of cell contact, intracellular ROS-dependent soluble factors, extracellular matrix, and apoptosis- associated signalling in MSCs co-culture contribute to hepatotrophic effect and HPc-induced potentiative effect. Co-transplantation with MSCs should improve therapeutic effects of HCT by enhancing survival and metabolism of co-transplanted hepatocytes.
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Benvenuti, Ticiana Thomazine. „Influência de células epiteliais mamárias malignas na expressão gênica de células estromais originárias de linfonodo axilar comprometido ou não comprometido de pacientes com câncer de mama“. Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-08072008-105031/.
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O desenvolvimento da metástase depende, entre outros fatores de um microambiente propício e é possível que as células do câncer de mama influenciem o perfil da expressão gênica das células estromais, tanto no tumor primário como no linfonodo regional. Para estudar este último evento obtivemos fibroblastos de linfonodos comprometidos (n=3) ou não (n=3) de pacientes com câncer de mama, os quais foram co-cultivados com células de câncer de mama MDA-MB-231, separados fisicamente por membranas porosas por 72 horas. O perfil da expressão gênica foi determinado utilizando-se uma plataforma de cDNA microarray. A presença de células MDA-MB231 modulou a expressão de 415 genes em fibroblastos de linfonodos não comprometidos e 779 genes em fibroblastos de linfonodos comprometidos, em relação a células mantidas em mono-cultura, sendo que 120 genes tiveram sua expressão reguladas em ambas comparações. A expressão destes genes determinou a separação dos fibroblastos mantidos em co-cultura daqueles mantidos isoladamente em cultura por análise de agrupamento hierárquico, indicando que a presença das células de câncer de mama influencia o perfil da expressão gênica das células. Entre as funções reguladas pela presença de células MDA-MB231 encontramse tradução e receptor de superfície ligado à transmissão de sinal, em fibroblastos de linfonodos não comprometidos e autofosforilação de proteína e ciclo celular, em fibroblastos de linfonodos comprometidos. Para determinar se a expressão gênica diferencial é um fenômeno que pode ser generalizado para o câncer de mama, realizamos ensaios utilizando outras amostras de fibroblastos obtidos de linfonodos comprometidos de 5 pacientes e de linfonodos não comprometidos de outras 5 pacientes (incluindo as 6 amostras de fibroblastos previamente analisadas), as quais foram co-cultivadas com três linhagens celulares de câncer de mama (MDA-MB231, MDA-MB435 e MCF-7). A expressão relativa de MAP2K3, AKAP8L e CREG1, inicialmente identificados como diferencialmente expressos em análise de cDNA microarray, foi então determinada por RT-PCR em tempo real. Observamos que nenhuma das três linhagens celulares influenciou a expressão de AKAP8L e CREG1 tanto em fibroblastos de linfonodos comprometidos quanto não comprometidos. MAP2K3 foi hiperexpresso tanto em fibroblastos de linfonodos comprometidos quanto em não comprometidos co-cultivados com células MDA-MB231, mas não com células MDA-MB435 ou MCF-7. Nossos resultados indicam que a presença de células MDA-MB231 influencia o perfil de expressão gênica de fibroblastos originário tanto de linfonodos comprometidos quanto de não comprometidos. Alguns genes comumente regulados em fibroblastos de linfonodo comprometido ou não comprometidos, incluindo MAP2K3, podem estar envolvidos no estabelecimento de metástase regional.Metastasis development may depend on a favorable microenvironment and breast cancer cells may influence stromal cells gene expression profile not only in the primary site but also in the lymph nodes. To study the latter event, fibroblasts were obtained from involved or uninvolved lymph nodes from six breast cancer patients and co-cultivated with breast cancer MDA-MB-231 cells, physically separated by porous membranes, for 72 hours. The gene expression profile was determined utilizing a cDNA microarray platform. The presence of MDA-MB231 cells modulated the expression of 415 genes in fibroblasts from uninvolved nodes and 779 genes in fibroblasts from involved nodes, as compared to fibroblasts cultured isolated, including 120 genes, which were regulated in fibroblasts from both origins. Unsupervised hierarchical clustering allowed a correct segregation of fibroblasts co-cultured with MDA-MB231 cells from fibroblasts cultured alone, indicating that the gene expression profile gene is influenced by the presence of breast cancer cells. Functions, which were regulated by presence of MDA-MB231 cells included translation and cell surface receptor linked signal transduction in fibroblasts from uninvolved nodes and protein autophosphorylation and cell cycle in fibroblasts from involved nodes. To determine whether this differential gene expression was a general process influenced by breast cancer cells, further assays were performed utilizing samples of fibroblasts from involved (n=5) and uninvolved nodes (n=5) from breast cancer patients (including the 6 samples of fibroblasts previously analyzed), which were co-cultured with three breast cancer cell lines (MDAMB231, MDA-MB435 and MCF-7). Relative expression of MAP2K3, AKAP8L and CREG1, initially identified as differentially expressed in co-cultured fibroblasts upon cDNA microarray analysis, was determined by RT-PCR real time. AKAP8L and CREG1 expression in fibroblasts from involved or uninvolved nodes was not influenced by the presence of any of the three cell lines. MAP2K3 was over-expressed in fibroblasts from both involved and uninvolved nodes when co-cultured with MDA-MB231 cells, but not with MDA-MB435 or MCF-7 cells. Our results indicate that the gene expression profile of fibroblasts from involved and uninvolved lymph nodes are influenced by the presence of MDA-MB231. Some genes commonly regulated in fibroblasts from both involved and uninvolved nodes, as MAP2K3, may be involved in regional metastasis establishment.
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Santos, Rosângela Portilho Costa. „Expressão gênica diferencial de fibroblastos de linfonodos comprometidos ou não comprometidos de pacientes com câncer de mama após cultura isolada ou co-cultura com células epiteliais mamárias normais ou malignas“. Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/5/5155/tde-19032007-145952/.
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As interações epitélio-mesênquima podem influenciar o desenvolvimento do tumor no sítio primário e no linfonodo comprometido de pacientes com câncer de mama. Nosso objetivo foi avaliar a taxa de proliferação e perfil gênico de fibroblastos de linfonodos comprometidos e não comprometidos obtidos de pacientes com câncer de mama e determinar a influência de células epiteliais mamárias normais (MCF10A) ou malignas (MDA-MB-231) na expressão gênica destes fibroblastos. Foram estabelecidas culturas primárias de fibroblastos de linfonodos (3 comprometidos e 3 não comprometidos) de 6 diferentes pacientes com câncer de mama e não houve diferença na taxa de proliferação destes fibroblastos. Co-culturas de células MCF10A ou MDA-MB-231 com fibroblastos, separadas fisicamente por membranas porosas, foram realizadas por 72 horas. O RNA total dos fibroblastos foi extraído, amplificado e o perfil gênico foi analisado usando-se uma lâmina de cDNA microarray. Fibroblastos de linfonodos comprometidos e não comprometidos apresentaram perfil gênico similar, pois apenas 13 genes foram modulados, sendo que maior expressão de PGBD3 e PTBP2 em fibroblastos de linfonodos comprometidos foi confirmada em ensaios de RT-PCR em tempo real. Em fibroblastos, a cocultura com células MCF10A alterou a expressão de maior número de genes que a co-cultura com células MDA-MB-231. Em fibroblastos originários de linfonodos não comprometidos mantidos em co-cultura com células MDA-MB-231 foram modulados 151 genes, em relação aos mesmos fibroblastos cultivados na ausência de células epiteliais, sendo que oito deles apresentaram variação de expressão superior a três vezes (BET1, ENTPD1, USP7, DAPK1, ERBB2 e NCF2). As células MDA-MB-231 modularam algumas vias de sinalização em fibroblastos não comprometidos, como vias do cálcio, insulina, hormônio liberador de gonadotrofina (GnRH) e da regulação do citoesqueleto de actina, mas o mesmo não ocorreu em fibroblastos de linfonodos comprometidos. Duzentos e quarenta e nove genes foram diferencialmente expressos em fibroblastos originários de linfonodos comprometidos mantidos em co-cultura com células MDA-MB-231 e quatro genes apresentaram variação superior a três vezes (ACLY, AXUD1, CLCN5 e PDE6D). Nestes fibroblastos, algumas funções biológicas foram reguladas apenas por influência da célula MDA-MB-231, entre as quais metabolismo de aminoácidos, excreção, transporte intra Golgi e regulação da forma celular. As vias de sinalização e funções biológicas reguladas em fibroblastos pela interação com células epiteliais malignas podem estar implicadas no desenvolvimento de metástases regionais no câncer de mama.Tumor development may be influenced by epithelial-mesenchimal interactions in the primary site as well as in the involved lymph node from breast cancer patients. Our aim was to evaluate the proliferation rate and gene profile of fibroblasts obtained from involved and uninvolved lymph nodes from breast câncer patients and to determine the influence of normal (MCF10A) or malignant (MDA-MB-231) mammary epithelial cells on the gene expression of these fibroblasts. Primary culture from fibroblasts obtained from 3 involved and 3 uninvolved lymph nodes (ILF and NILF) from 6 different breast cancer patients was established and no difference in their proliferation rate was detected. These fibroblasts were co-cultured with MCF10A or MDA-MB-231 cells, physically separated by a porous membrane for 72 hours. Total RNA was extracted from the cells, amplified and the gene profile from fibroblasts cultured alone or with epithelial cells was analyzed by cDNA microarray slides. Fibroblasts from involved and uninvolved lymph nodes present a similar gene profile as only 13 genes were differentially expressed between them, including PGBD3 and PTBP2, which higher expression in fibroblasts from involved lymph nodes was confirmed by real time RT-PCR. In fibroblasts, more genes seem to be regulated upon co-cultured with MCF10A than with MDA-MB-231 cells. In fibroblasts from uninvolved lymph nodes co-cultured with MDA-MB-231 cells, 151 genes were modulated as compared with fibroblasts cultured alone, and eight of them, presented an expression variation superior to three times (BET1, ENTPD1, USP7, DAPK1, ERBB2 e NCF2). In these fibroblasts, MDA-MB-231 could modulate some signaling pathways as calcium, insulin, gonadotrophin releasing hormone (GnRH) and actin cytoskeleton regulation signaling pathways. Two hundred forty nine genes were differentially expressed by fibroblasts from involved lymph nodes co-cultured with MDA-MB-231 cells, four of which with an expression variation superior to three times (ACLY, AXUD1, CLCN5 e PDE6D). In the last condition, fibroblasts had some biological functions regulated upon MDA-MB-231 cells influence, among which amino acid metabolism, excretion, intra-Golgi transport and regulation of cell shape. Signaling pathways and biological functions regulated in fibroblasts after interaction with malignant epithelial cells might be implicated in the development of regional metastasis in breast cancer.
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Springer, Marco [Verfasser]. „Effects of the regulators of pigmentation 3-isobutyl-1-methylxanthine, kojic acid and arbutin on newly developed cocultures and skin equivalents composed of HaCaT cells and human melanocytes / Marco Springer“. Aachen : Shaker, 2004. http://d-nb.info/1170530249/34.
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Muhr, Christian [Verfasser], Achim [Akademischer Betreuer] Göpferich und Torsten [Akademischer Betreuer] Blunk. „Establishment and characterization of a human 3-D fat model - Adipogenesis of hASC in a spheroid model; 3-D cocultures of adipocytes and endothelial cells / Christian Muhr. Betreuer: Achim Göpferich ; Torsten Blunk“. Regensburg : Universitätsbibliothek Regensburg, 2013. http://d-nb.info/1043631380/34.
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Joshi, Jyotsna. „Engineering the Micro-Environment Niche of Human Bone Marrow-Derived Mesenchymal Stem Cells for Enhanced Cardiac Tissue Regeneration“. Cleveland State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=csu1543925924170549.
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Seto, Song P. „The development of heparin-based materials for tissue engineering applications to treat rotator cuff tendon injuries“. Diss., Georgia Institute of Technology, 2014. http://hdl.handle.net/1853/51898.
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Surgical repair of torn rotator cuff tendons have a high rate of failure and does not address the underlying pathophysiology. Tissue engineering strategies, employing the use of multipotent progenitor cells or growth factors, represent potential therapies to improve the outcome of rotator cuff surgery. The use of glycosaminoglycan-based biomaterials in these therapies may enhance the effectiveness of cell and growth factor delivery techniques. Furthermore, understanding the cellular and molecular mediators in tendon overuse can help elucidate the causes of tendon degeneration. Thus the overall goals of this dissertation were to 1) develop heparin-based biomaterials to enhance cell pre-culture and maintain growth factor bioactivity and 2) characterize the histological and enzymatic changes in a supraspinatus tendon overuse model. To investigate the use of heparin in enhancing dynamic signaling, mesenchymal stem cells (MSCs) were encapsulated in heparin-containing hydrogels and evaluated for differentiation markers when cocultured with a small population of differentiated cells. To probe the effect of sulfation of heparin on the interactions with protein, selectively desulfated heparin species were synthesized and evaluated for their ability to bind and protect proteins. Finally, to develop a tendon overuse model that can become a test bed for testing future targeted therapeutics, an animal model was evaluated for tissue damage and protease activity. Together these studies represent a multi-pronged approach to understanding how tendon tissues become degenerative and for developing technologies to improve the biological fixation of tendon to bone in order to reduce the need for revision surgeries.
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„Microbial Electrochemical Cells for Selective Enrichment and Characterization of Photosynthetic and Haloalkaliphilic Anode-Respiring Bacteria“. Doctoral diss., 2013. http://hdl.handle.net/2286/R.I.18043.
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abstract: Microbial electrochemical cells (MXCs) are promising platforms for bioenergy production from renewable resources. In these systems, specialized anode-respiring bacteria (ARB) deliver electrons from oxidation of organic substrates to the anode of an MXC. While much progress has been made in understanding the microbiology, physiology, and electrochemistry of well-studied model ARB such as Geobacter and Shewanella, tremendous potential exists for MXCs as microbiological platforms for exploring novel ARB. This dissertation introduces approaches for selective enrichment and characterization of phototrophic, halophilic, and alkaliphilic ARB. An enrichment scheme based on manipulation of poised anode potential, light, and nutrient availability led to current generation that responded negatively to light. Analysis of phototrophically enriched communities suggested essential roles for green sulfur bacteria and halophilic ARB in electricity generation. Reconstruction of light-responsive current generation could be successfully achieved using cocultures of anode-respiring Geobacter and phototrophic Chlorobium isolated from the MXC enrichments. Experiments lacking exogenously supplied organic electron donors indicated that Geobacter could produce a measurable current from stored photosynthate in the dark. Community analysis of phototrophic enrichments also identified members of the novel genus Geoalkalibacter as potential ARB. Electrochemical characterization of two haloalkaliphilic, non-phototrophic Geoalkalibacter spp. showed that these bacteria were in fact capable of producing high current densities (4-8 A/m2) and using higher organic substrates under saline or alkaline conditions. The success of these selective enrichment approaches and community analyses in identifying and understanding novel ARB capabilities invites further use of MXCs as robust platforms for fundamental microbiological investigations.Dissertation/Thesis
Ph.D. Microbiology 2013
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Hassan, E., P. Deshpande, F. Claeyssens, Stephen Rimmer und S. MacNeil. „Amine functional hydrogels as selective substrates for corneal epithelialization“. 2014. http://hdl.handle.net/10454/10459.
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NoThe aim of this study was to develop a synthetic hydrogel to act as a corneal substitute capable of selectively supporting the adhesion and proliferation of limbal epithelial cells (LECs) while inhibiting growth of limbal fibroblasts. Deficiency of LECs causes conjunctival epithelial cells to move over the cornea, producing a thick scar pannus. Unilateral defects can be treated using LEC cultured from the unaffected eye, transplanting them to the affected cornea after scar tissue is removed. The underlying wound bed is often damaged, however, hence the need to develop a corneal inlay to aid in corneal re-epithelialization. Transparent epoxy-functional polymethacrylate networks were synthesized using a combination of glycerol monomethacrylate, ethylene glycol dimethacrylate, lauryl methacrylate and glycidyl methacrylate that produced two different bulk hydrogel compositions with different equilibrium water contents (EWCs): Base 1 and Base 2, EWC=55% and 35%, respectively. Two sets of amine-functional hydrogels were produced following reaction of the epoxide groups with excesses of either ammonia, 1,2-diamino ethane, 1,3-diamino propane, 1,4-diamino butane or 1,6-diamino hexane. Neither series of hydrogels supported the proliferation of limbal fibroblasts irrespective of amine functionalization but they both supported the adhesion and proliferation of limbal epithelial cells, particularly when functionalized with 1,4-diamino butane. With Base 1 hydrogels (less so with Base 2) a vigorous epithelial outgrowth was seen from small limbal explants and a confluent epithelial layer was achieved in vitro within 6days. The data support the development of hydrogels specific for epithelial formation.
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Lohaus, Raphaela. „Etablierung der Organotypischen Hirnschnitt-Kokultur als Tumor-Invasionsmodell“. Doctoral thesis, 2013. http://hdl.handle.net/11858/00-1735-0000-000D-FB55-A.
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39
Jing, Duohui. „Mobilisation, Isolation and Coculture of Haematopoietic Stem Cells“. Doctoral thesis, 2010. https://tud.qucosa.de/id/qucosa%3A25373.
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Since decades, hematopoietic stem cell transplantation (HSCT) has become a well established treatment modality for hematological malignancies and non-malignant disorders. Autologous and allogeneic hematopoietic stem cells (HSCs) mobilized into the peripheral blood (PB) have been used as a preferred source of transplantable stem cells1-3. And umbilical cord blood (UCB) has been introduced as a more attractive HSC source for HSCT, because fetal stem cells in UCB are speculated to be more primitive in comparison to adult stem cells. However the limited amount of HSCs is limiting their application for stem cell therapy in clinic. Therefore, people started to utilize extra-embryonic tissue to harvest more fetal stem cells, while people also tried to optimize the clinical protocol to mobilize more adult stem cells out of adult bone marrow. The innovative strategies and feasible procedures were discussed in this thesis.
The axis of the chemokine receptor CXCR4 and its ligand SDF-1 is important for trafficking and homing of HSCs. It has already been demonstrated that the bicyclam AMD3100, a CXCR4 antagonist, in combination with G-CSF is able to induce a significant mobilization of CD34+ cells4. And human placenta is a potent hematopoietic niche containing hematopoietic stem and progenitor cells throughout development5. The homing of HSCs to the placenta is probably also mediated by the expression of SDF-1 as demonstrated for the bone marrow niche. In this study (part 1 of the chapter “Results and discussions”), we utilized AMD3100 to mobilize HSCs from placenta. And we can demonstrate that the CXCR4 antagonist AMD3100 mobilise placenta derived CD34+ cells ex utero already after 30 min of incubation and may further enhance the efficacy of harvesting placenta-derived HSC.
The alpha4 integrin CD49d is involved in migration and homing of hematopoietic stem cells (HSC). Therapeutic application of natalizumab, an anti-CD49d antibody, in patients with multiple sclerosis (MS) has been associated with increased levels of circulating CD34+ progenitors. In our study (part 2 of the chapter “Results and discussions”), we compared circulating HSCs from MS patients after natalizumab treatment and HSCs mobilized by G-CSF in healthy volunteers, with regard to their migratory potential, clonogenicity and gene expression. CD34+ cells in the blood and marrow of natalizumab-treated patients expressed less of the stem cell marker CD133, were enriched for erythroid progenitors (CFU-E) and expressed lower levels of adhesion molecules. The level of surface CXCR-4 expression on CD34+ cells from patients treated with natalizumab was higher compared to that of CD34+ cells mobilized by granulocyte-colony stimulating factor (G-CSF) (median 43.9% vs. 15.1%). This was associated with a more than doubled migration capacity towards a chemokine stimulus. Furthermore, CD34+ cells mobilized by natalizumab contained more m-RNA for p21 and less MMP9 compared to G-CSF mobilised HSC. Our data indicate that G-CSF and CD49d blockade mobilize different HSC subsets and suggest that both strategies may be differentially applied in specific cell therapy approaches.
In order to further improve the clinical outcome of HSC transplantation, many groups are focusing on ex vivo maintain or expand HSC. Unfortunately, the maintenance of HSC in vitro is difficult to achieve because of their differentiation. This is presumably caused by a lack of appropriate cues that are provided in vivo by the microenvironment. Indeed, HSCs located in the bone marrow are interacting with a specific microenvironment referred to as the stem cell niche, which regulates their fate in terms of quiescence, self-renewal and differentiation. An orchestra of signals mediated by soluble factors and/or cell-to-cell contact keeps the balance and homeostasis of self-renewal, proliferation and differentiation in vivo. To investigate the communication between HSCs and the niche, coculture assays with mesenchymal stromal cells (MSCs) were performed in vitro. Here, we can demonstrate that cell-to-cell contact has a significant impact on hematopoietic stem cells expansion, migratory potential and stemness. In this study (part 3 of the chapter “Results and discussions”), we investigated in more detail the spatial relationship between hematopoietic stem cells and mesenchymal stromal cells during ex-vivo expansion. And we defined three distinct localizations of HSCs relative to MSC layer: (i) those in supernatant (non-adherent cells); (ii) cells adhering on the surface of mesenchymal stromal cells (phase-bright cells) and (iii) cells beneath the mesenchymal stromal cells (phase-dim cells). Our data suggest that the mesenchymal stromal cell surface is the dominant location where hematopoietic stem cells proliferate, whereas the compartment beneath the mesenchymal stromal cell layer seems to be mimicking the stem cell niche for more immature cells. Our data provide novel insight into the construction and function of three-dimensional HSC–MSC microenvironments.
In summary, we provided a new method to isolate fetal stem cells from extra-embryonic tissue (i.e. placenta) in the first part, then we discussed an innovative strategy with CD49d blockade to improve clinical modality for adult stem cell mobilization in the second part, and finally we investigated HSC maintenance and expansion in vitro and provided feasible way to mimic HSC niche in vitro in the last part.
This thesis contributes to HSC-based stem cell therapy in two aspects, i.e. 1) fetal and adult stem cell isolation holding great therapeutic potential for blood diseases; 2) ex vivo stem cell manipulation providing a valuable platform to model HSC niche regulation.
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Bach, Martin. „Der Einfluss muriner mesenchymaler Stammzellen auf murine zytokin induzierte Killerzellen in der Kokultur“. Doctoral thesis, 2013. https://ul.qucosa.de/id/qucosa%3A12814.
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Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required.
In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.:Inhaltsverzeichnis I
Abbildungsverzeichnis III
Tabellenverzeichnis IV
Bibliographische Beschreibung V
Abkürzungsverzeichnis VII
1 Einleitung 1
1.1 CIK-Zellen (CIK) 3
1.1.1 Merkmale von CIK-Zellen 3
1.1.2 Wirkungsmechanismen von CIK-Zellen 3
1.1.3 Studienlage 4
1.1.4 Bisherige Ansätze zur Verbesserung der Kultivierungsbedingungen 6
1.2 Mesenchymale Stammzellen (MSC) 7
1.2.1 Allgemein 7
1.2.2 Differenzierung von MSC 7
1.2.3 Heterogenität und Einflussfaktoren der MSC - Identitätsproblematik 8
1.2.4 Charakterisierung von MSC 9
1.2.5 Therapeutische Einsatzmöglichkeiten von MSC 11
2 Zielformulierung 15
3 Material und Methoden 16
3.1 Tiere 16
3.2 Materialien 17
3.2.1 Materialien für Zellkultur 17
3.2.2 Materialien für FACS-Analyse 18
3.2.3 Materialien für Zytotoxizitätsassay 19
3.2.4 Materialien für CFU-F-Assay 20
3.3 Methoden 21
3.3.1 Statistische Auswertung 21
3.3.2 Zellkultur 22
3.3.3 FACS (Fluorescence Activated Cell Sorting) 26
3.3.4 Markierung der MSC mit DiD 28
3.3.5 Zytotoxizitätsassay (LDH-Freisetzungsassay) 29
3.3.6 CFU-F-Assay 32
4 Ergebnisse 34
4.1 Beeinflussung der Wachstumskurve 34
4.1.1 Der Wachstumskurvenverlauf von CIK-Zellen (Kontrollen) 34
4.1.2 Der Wachstumskurvenverlauf von CIK-Zellen in der Kokultur mit MSC 35
4.1.3 Der Wachstumskurvenverlauf in MSC-konditioniertem Medium 37
4.1.4 Der Wachstumskurvenverlauf bei Restimulierung an Tag 14 38
4.2 Beeinflussung des Oberflächenphänotyps 40
4.2.1 Der Oberflächenphänotyp von CIK-Zellen 40
4.2.2 Vergleich Oberflächenphänotyp Kontrollen mit kokultivierten CIK 43
4.3 Beeinflussung der Vitalität 46
4.4 Beeinflussung der Zytotoxizität 48
4.5 Identifizierung der MSC 49
4.5.1 Adhärenz an Plastikoberflächen 50
4.5.2 Fibroblastenähnliche Wachstumsmorphologie 50
4.5.3 Wachstum in Colony-Forming-Units 51
4.5.4 Der Oberflächenphänotyp von MSC 53
4.6 Schicksal der MSC in der Kokultur 54
4.6.1 Der Oberflächenphänotyp der adhärenten Zellen nach Kokultur 54
4.6.2 Kokultur mit DiD gelabelten MSC 57
5 Diskussion 59
5.1 Beeinflussung der Wachstumskurve 60
5.1.1 Mechanismen der Beeinflussung des Wachstumskurvenverlaufs 60
5.1.2 Fehlerbetrachtung 68
5.2 Identifizierung der CIK sowie Beeinflussung von Phänotyp und Vitalität 69
5.3 Beeinflussung der Zytotoxizität 70
5.3.1 Vergleich Zytotoxizität Kontrollen mit Kokulturen 70
5.3.2 Fehlerbetrachtung 71
5.4 Identifizierung der MSC 72
6 Schlussfolgerung 75
7 Ausblick 77
8 Zusammenfassung 79
Literaturverzeichnis 83
Danksagung I
41
Gómez, Aristizábal Alejandro. „Human Umbilical Cord Perivascular Cells: Putative Stromal Cells for Hepatocytes“. Thesis, 2012. http://hdl.handle.net/1807/32722.
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Liver failure, which is the result of liver injury and pathological inflammation, is currently only successfully treated by organ transplantation. However donor organ shortages preclude transplantation for many patients in need. Thus, bioartificial liver systems (BALS) are being developed as a bridge to transplantation, or to create an environment conducive to liver regeneration. Hepatocytes, the main functional cells of the liver, are the cells of choice for BALSs, but in standard conditions ex vivo, they rapidly suffer from a reduction of their functionality and viability. Coculture with stromal cells, for example bone marrow mesenchymal stromal cells (BM-MSC), has been shown to improve, and extend, hepatocyte function ex vivo up to 21 days. But, only small numbers of BM-MSCs can be harvested from adult volunteers. We have previously described an alternative, more plentiful, source of MSCs — human umbilical cord perivascular cells (HUCPVC) — that are easily expanded and non-alloreactive. Our hypothesis was that HUCPVCs are putative stromal cells for hepatocytes. Our results show that HUCPVCs improved hepatocyte albumin secretion, urea synthesis and maintained hepatocyte cytochrome activity and the expression of hepato-specific genes. Furthermore, there was a net proliferation of hepatocytes, which were polarized in coculture with HUCPVCs, as judged by functional bile canaliculi that were present for up to 40 days. We found that both soluble and non-soluble factors contributed to these effects, while neither was able to allow net proliferation individually. Moreover, HUCPVCs expressed both hepato-trophic and anti-inflammatory factors, at different levels to BM-MSCs, indicating the potential for differential hepato-therapeutics. We conclude that HUCPVCs are putative stromal cells for hepatocytes; they improve hepatocyte functionality, polarity, morphology and net proliferation, and thus present an opportunity for the improvement of both BALS function and liver therapy.
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Chou, Ya-Shuan, und 鄒亞璇. „Tissue Engineering of Human Salivary Gland: Stem Cells and Coculture System“. Thesis, 2015. http://ndltd.ncl.edu.tw/handle/yp8743.
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博士國立臺灣大學
醫學工程學研究所
103
Salivary gland is an exocrine gland that is responsible for saliva production. The secretion of saliva contains digestive enzymes, growth factors, and antimicrobial agents. This function is crucial in the processes of digestion, lubrication, and protection in the body. Therefore, salivary gland hypofunction can result serious negative effects on the patients quality of life. Radiation therapy (RT) for head and neck cancer, and autoimmune diseases (such as Sjogren’s syndrome) can cause unavoidable coirradiation of surrounding normal tissues such as the salivary glands. This effect results in irreversible damage of salivary glands hence to significantly reduces salivary output. Salivary gland hypofunction and xerostomia lead to hindering of speech (dysphonia), difficulty of swallowing (dysphagia), influences on nutrition (dysnutritia) and others. Currently, there is no treatment available to permanently treat dysfunctional salivary glands. Constructing artificial salivary glands with tissue engineering may be a viable curative procedure to treat patients suffering from dry mouth. Tissue engineering applications in salivary glands require a significant amount of salivary gland cells and an understanding of gland cell-related coordination and function. Many experimental induce the increase of salivary gland stem/progenitor cells with duct ligation or fluorescence-activated cell sorting by antibodies. This research establishes a simple and highly reproducible protocol for isolation and characterization of stem/progenitor cells obtained from human salivary glands. The percentage of CD49f and CD90 double-positive cells was increased at 14 days after seeding, and then decreased at 28 days. The gene expression levels of ALDH1A1 and ALDH1A3 were also at the highest at 14 days after seeding. Cultured cells can successfully differentiate into adipocytes and osteocytes. E-cadherin expression in cultured cells increased with time while vimentin expression gradually decreased to a very low level after prolonged culture, inferring a MET during the repopulation process of cell culture. Our study confirmed the existence of progenitor cells in cultured adult human parotid glands. This research was also established a simple culture method to obtain sufficient progenitor cells for further tissue engineering studies. However, maintaining the expression levels of amylase in culturing salivary gland epithelial cells is important.Salivary gland cells in vivo are surrounded by a complex stromal environment, in which fibroblasts are the main cell type in proximity to the gland cells. An appropriate number of fibroblasts in contact with the parotid gland acinar cells (PGACs) is necessary to promote PGAC function. Fibroblast-secreted bFGF may play a paracrine signaling role in the regulation of α-amylase expression in PGACs. Interestingly, the effect of fibroblast conditioned medium from PVDF on the α-amylase expression of PGACs was obviously enhanced. By growth factor protein array assay, higher NT-4 expression was observed in PVDF-derived fibroblast conditioned medium. Fibroblasts might be reprogrammed into neural-like cells after cultured on appropriate biomaterials. In these studies, the interaction between PGACs, fibroblasts, and biomaterials was investigated. This could be treated patients with xerostomia and applying directly by re-implantation of autologous salivary gland tissue engineering. The re-implantation treatment can be a major contribution to their quality of life.
43
Jing, Duohui [Verfasser]. „Mobilisation, isolation and coculture of haematopoietic stem cells / vorgelegt von Duohui Jing“. 2009. http://d-nb.info/1013557603/34.
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44
Yueh, Ting-Ju, und 岳庭如. „Automated embryo trapping and coculture of endometrial cells within a microfluidic device“. Thesis, 2013. http://ndltd.ncl.edu.tw/handle/82037322222412309826.
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碩士國立清華大學
動力機械工程學系
101
One in six couples worldwide has difficulty conceiving children in 2013. People have been suffering from infertility for the past two decades. In this research, Reproductive technology has been applied for the goal of improving the low pregnancy rate issues by integrating both microfluidic techniques and coculture of endometrial cells into enhance the embryo development in vitro. Traditional methods towards embryo development in vitro still required the complicated procedures which were all done manually for position the embryo, tracking each development, replacing medium and moving the embryo. The labor operation might increase the risks of cell damage. In this study, the comparison of embryo coculture with endometrial cells on chip and in dish was investigated. This master study proposed a design of an automated positioning single murine embryo by utilizing a hydraulic concept and electrical circuit analysis to reduce the operation procedure and adverse effects on organism. The coculture of embryo with endometrial cells within a dynamic perfusion system was also applied in this master study to mimic the embryo development in vivo. The dynamic perfusion system was performed to exclude the waste, provide fresh medium and be combined with the designed microchannels of specific microfluidic streamline in the coculture chamber to minimize the trapped bubbles to enhance the coculture environment in vitro for murine embryo culture. The automated embryo coculture device has been achieved in this study for the management of individual mammalian embryos by using the dynamic microarray format. The developed microsystem can manage and coculture individual embryos in each microchamber. The embryo development in whole culture period could be tracked via this microsystem design. 8-cell-stage murine embryos were used for the starting stage of embryo developments in our experiments. The results showed that the Blastocyst development rates in traditional method and device are 55.6±3.4% and 61.8±3.3%, respectively, for the monoculture group. They are 69.8±7.8% and 77.7±6.7%, respectively, for the coculture group. In addition, the microfluidic device developed in this master research is simple in operation, and is feasible to achieve the higher blastocyst rates. The coculture platform mimics the micro environment of embryo growth in vivo to enhance its overall development. An automated prototype is achieved through this master study. The development of individual embryo could be enhanced via this microsystem, which is a very promising coculture platform in vitro.
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Ziegler, Elke. „Characterization of human breast cancer cells affected by coculture conditions and kisspeptin-10“. Doctoral thesis, 2013. http://hdl.handle.net/11858/00-1735-0000-0001-BAA6-8.
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46
Tsai, Ching-Wen, und 蔡靜雯. „The Effect of Chitosan Treatment on Coculture System of Cancer Cells and Senescence-Delaying of Fibroblasts“. Thesis, 2017. http://ndltd.ncl.edu.tw/handle/qavbpd.
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博士國立臺灣大學
醫學工程學研究所
106
An ideal in vitro drug screening model is important for the drug development. In addition to monoculture systems, coculture systems have been used to mimic the in vivo tumor tissues because cell-cell and cell-extracellular matrix interactions can be studied. In this study, suspension core/shell coculture multicellular spheroids on chitosan are developed. Based on the characteristic of chitosan inhibiting cell adhesion, SW620 (colon cancer cell line), 3A6 (mesenchymal stem-like cell line) and Hs68 (foreskin fibroblast line) cells can aggregate to form 3D coculture spheroids with intimate cell contacts. CD44 is extensively expressed within adult bone marrow and has been considered as an important marker for cancer stem cells in some types of tumors. Therefore, it is used to understand the variations of stem cells and cancer cells when cells culturing on chitosan firstly. When cells are cocultured on chitosan, 3A6 and Hs68 cells always locate in the core of spheroids and are completely enveloped by SW620 cells following the differential adhesion hypothesis. Moreover, the core cells can stimulate the shell SW620 cells to enhance activity and resistance against the cytotoxicity effect of chemotherapy drugs. Therefore, based on the specificity of the core/shell coculture multicellular spheroids, a novel in vitro tumor model is proposed. Fibroblasts have been extensively used as a model to study cellular senescence. The second purpose of this study is to investigate whether the human foreskin fibroblast aging process can be regulated by using the chitosan. Senescent cells are collected and seeded on chitosan to form multicellular spheroids. The protein expression of senescence-associated secretory phenotypes (SASPs) and senescence-associated molecular markers of these cells in multicellular spheroids are downregulated significantly. Following chitosan treatment, fibroblasts reseed on TCPS show lower SA β-gal activity, and higher cellular motility and proliferation ability. Cells can form suspending multicellular spheroids on many biomaterials, but only chitosan is capable of delaying senescence of fibroblasts. Therefore, in addition to the structure of multicellular spheroids, chitosan itself should play an important role in delaying fibroblast senescence. In addition to the intracellular TGF-β expression, the extracellular TGF-β expression is also downregulated by chitosan. TGF-β signaling pathway is involved in the chitosan-mediating fibroblast senescence process. Finally, whether the senescence-delaying effect of chitosan can be applied to other cells, such as human synovial membrane derived cells (SCs) and anterior cruciate ligament fibroblasts (ACLs) is examined. From the studied data, we find that chitosan not only delays the senescence but also enhances the functions of SCs and ACLs, which is benefit for chitosan applying on the cell therapy.
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Tsai, Ming-Tzu, und 蔡明慈. „Effects of Different Stimulation Time of sPEMF on Apoptosis of Osteoclast-Like Cells Developed from Coculture of Osteoblast Cells and Bone Marrow Cells“. Thesis, 2002. http://ndltd.ncl.edu.tw/handle/81046127433681073592.
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碩士中原大學
醫學工程研究所
90
Osteoporosis occurs when the activity of bone resorption is larger than the activity of bone formation. The osteoclast is one of the most direct and powerful factors, which is involved in bone resorption. Our lab have been proved that pulsed electromagnetic fields (PEMFs) with specific parameters influence the inhibition of osteoclast differentiation and formation, and this study investigates the effect of PEMF on osteoclast apoptosis. First, a novel coculture system is established to develop osteoclast-like cells (OCLs), which are exposed to PEMFs for different stimulation times, and OCL apoptosis rates are assayed at different times after PEMF exposure. We expect that PEMFs have effects on treatment or prevention of osteoporosis by inducing osteoclast apoptosis. In this study, bone marrow cells (BMCs) obtained from 10~12 -month-old adult female Wistar rats were the major cells in this coculture system. Osteoblasts (OB) were obtained from new-born ICR mice and new-born Wistar rats, and were individually cocultured with BMCs. OB density was 1x106 cells/6-cm dish, and BMC densities were 2x107, 4x107, and 8x107 cells/6-cm dish, giving a total 6 of types of coculture models. 10 nM 1α, 25(OH)2D3 was added in culture medium in coculture periods to enhance OCL differentiation. On the eighth day, purified OCLs were acquired after isolation by 0.15% collagenase, and isolated OCLs were exposed to PEMFs with different stimulation times. There were four groups: Control, PEMF-1 (exposure for 1 hr ), PEMF-8 (exposure for 8 hr), and PEMF-16 (exposure for 16 hr). The PEMF parameters were: single pulse, 7.5 Hz of frequency, and 2 mV/cm of induced electric field intensity. Apoptosis rates of OCLs were assayed by PI-stain method individually at 0, 24, 48, and 72 hr after PEMF exposure, where the time point after isolation was defined as 0 hr. For qualitative analysis of OCLs there were two processes. In one, Tartrate-resistant acid phosphatase (TRAP) was used as a marker of acid phosphatase of OCLs, and TRAP-positive multinucleated (more than 3 nuclei) cells (TRAP-PMCs) were identified as OCLs. In the other, bone resorption pits on the surface of porcine cortical bone slices formed by OCLs were examined by scanning electron microscope (SEM). The results showed that: (1) Numerous OCLs (TRAP-PMCs) were observed in an optimal coculture system established by OB (1x106 cells/6-cm dish) of new-born mice and BMCs (2x107 cells/6-cm dish) of adult female Wistar rats. However, the other five coculture models failed to develop TRAP-PMCs. Considering the uniformity of PEMFs, a 3.5-cm dish was substituted for the 6-cm dish and the optimal cell densities of OB and BMCs subsequently change into 4x105 and 2x107 respectively, and numerous TRAP-PMCs still appeared in this coculture system. (2) The results of apoptosis assay revealed that the rate of apoptotic OCLs increased when the apoptosis assay time increased, reaching the highest rate of 98%. Nuclear fragments of apoptotic OCLs appeared clearly after a longer time period after isolation. Compared with PEMF-1 and Control groups, the rates of apoptotic OCLs between 0~72 hr were similar. Compared with Control, the rate of apoptotic OCLs in PEMF-8 and PEMF-16 were both higher at 48 hr of apoptosis assay time points, and others were similar at 0, 24, and 72 hr of apoptosis assay time points. It suggested that both PEMF stimulation time and apoptosis assay time points are important roles in inducing OCL apoptosis by PEMFs with specific parameters. The rate of apoptotic OCLs increased at 48 hr after exposure to PEMFs for 8 and 16 hr.
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Chen, Chi-Fan, und 陳紀帆. „A Microfluidic Device for the Automatic Trapping of Embryos and the Coculture with Stromal Cells in Vitro“. Thesis, 2014. http://ndltd.ncl.edu.tw/handle/p628b7.
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碩士國立清華大學
動力機械工程學系
102
Infertility is a worldwide important issue. Assisted Reproductive Technology (ART) becomes one of the most important studies in the 21st Century. The reasons of infertility are most from the modern plague, such as poor nutrition, obesity, alcohol, cigarette, stress and drugs. Recently, in vitro fertilization (IVF), which holds the highest success rate, is a major treatment for infertility. In this research, the microfluidic devices made by a simple fabrication were developed. We provided the method and operation by integrating both microfluidic techniques and coculture of stromal cells with embryos to mimic the uterus in vitro. The culturing by the dynamic system with the fresh medium also enhances the embryo development. Furthermore, the embryos were automatically and individually trapped via the flow resistance design. The design was simulated and analyzed via the commercial software, CFD and ANSYS CFX, to optimize the flow channel design as well as reduce the manual operation, which resulted in the possibility of embryos damaged. And the most important thing is that the chip is easy to track and manage individual embryos. The integrated Labchip functions include automatic trapping, dynamic perfusion and coculture to mimic the uterus in vitro. The experimental results show that this Labchip could provide better environment compared with some different conditions such as monoculture and static culture. The embryos could be cultured in comparable blastocyst rate versus the traditional method (60% vs. 57.8%). Besides, the embryos cultured on the chip have the faster growth rate than the traditional method. All of these mean that our Labchip provides a bionic environment for embryo developing. Finally, we transfer the embryos cultured from our Labchip into the maternal uterus and born the mice. It verifies the feasibility and prospective of this Labchip.
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Chao, Hsiao-Huei, und 趙小慧. „Differentiation into Hepatocytes and Maintenance of Hepatocyte Functions by Bone Marrow Stromal Cells Coculture with Injured Hepatocytes“. Thesis, 2008. http://ndltd.ncl.edu.tw/handle/50920488582468215317.
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碩士中國醫藥大學
臨床醫學研究所碩士班
96
Liver transplantation is the gold standard treatment for end-stage liver failure and for numerous liver based inborn errors of metabolism. However, organ shortage remains a major limiting factor and alternative solutions are being examined in the liver therapy field. Liver cell transplantation has become the most promising alternative. Compared to liver transplantation, this procedure is less invasive, less expensive, and fully reversible. But it is still limited by cell viability, modest engraftment and tissue availability. Increasing interest is carried to stem cells regarding the recent demonstration of their plasticity. Theoretical advantages of mesenchymal stem cells for tissue regenerative medicine are multiple: ease of harvest, proliferation capacity, efficiency of in vitro transfection and potential use of autologous cells. Despite encouraging results, key pitfalls remain while using stem cells-derived hepatocyte-like cells: low engraftment rate and no strong liver repopulation level in animal models. To identify the differentiation plasticity of adult bone marrow mesenchymal stem cells (MSCs) into hepatocyte-like phenotypes, we used a co-culture model with hepatocytes. Furthermore, we investigated whether MSCs can protect the acutely injured hepatocytes, stimulate regeneration and restore the functions of hepatocytes. This data have evidenced that the guided hepatic differentiation of MSCs is proportionate to the activity of co-cultured hepatocytes. The hepatogenic environment is crucial to MSCs differentiation. It evidenced the trans-differentiation potential of MSCs developing to the hepatocytes and restoration of the functions of acutely injured hepatocytes. Further future therapeutic application in hepatic regeneration will be focused on the created imitated niches for MSC to maintain the survival and functions of hepatocytes.
50
Chen, Lijuan, und 陳儷娟. „The Effects of Coculture of Rhodotorula glutinis and Scenedesmus obliquus on Cells Growth and Total Lipids Accumulation“. Thesis, 2013. http://ndltd.ncl.edu.tw/handle/90987711777135098596.
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碩士東海大學
化學工程與材料工程學系
101
During cultivation, Rhodotorula glutinis utilized C sorce and released CO2. Moreover, organic acids were synthesized and the pH of culture dropped. As a result, the growth of yeast was inhibited. Scenedesmus obliquus utilized CO2 and released O2 during photosynthesis. The cells growth of R. glutinis and S. obliquus would be raised with light exposed. The metabolites are inhibiative to R. glutinis and S. obliquus in the medium were removed and it was sutable to grow for yeast and algae. The accumulations of biomass and total lipids in the coculture are suitable for biodiesel production. The biomass concentration of 11.1 ± 0.44 g/L and the total lipids accumulation of 1.777 ± 0.278 g/L were obtained by R. glutinis in the pure culture. The biomass concentration of 2.0 ± 0.06 g/L and the total lipids accumulation of 0.370 ± 0.015 g/L were obtained by S. obliquus in the pure culture. The biomass concentration of 14.05 ± 0.07 g/L and the total lipids accumulation of 3.594 ± 0.251 g/L were obtained in the coculture. It was shown the results of the coculture are better than the pure culture’s. The total percentages of palmitic (C16：0) and oleic acids (C18：1) are raised to 90.99 % of the couclutre from the 84.68 % of R. glutinis and 83.14 % of S. obliquus in the pure culture. The cells growth and total lipids accumulation are effected by the composition of the medium and the environment. The ratio of inoculation of yeast and algae, the concentration of N source, the concentration of glucose, NaCl, initial pH, the baffle, the temperature and the intensity of light were studied in flaskes. The possipility of scale-up was studied in the 5 L fermentor and 6 L air-lift bioreactor. The effect of the ratio was unobvious based on the experiment of the ratio of inoculation. Based on N source experiment, the algae prefered inorganic N source (KNO3) and the cells growth were raised with concentration of KNO3 increasing. Ferthermore, the yeast prefered organic N source (Yeast extract, YE). Compared with algae, the cells growth of yeast was progressed better. The higher biomass and total lipids accumulation were obtained from yeast. Under N-limited, the total lipids accumulation is promoted with glucose concentration increased. The biomass of 15.1 ± 1.04 g/L, the total lipids accumulation of 5.952 ± 0.392 g/L and the total lipid content of 39.34 ± 1.05 % are obtained at 50 g/L glucose. The total lipids accumulation and the yield of lutein were advanced with NaCl adding. The total lipids accumulation was increased 13.4 %. The yield of β-carotene was increased 435 ~ 490 % with the addition of 0.5 g/L NaCl delayed. The growth of algae was inhibited at high pH based on the pH experiment. The growth of yeast was advanced when the aeration was increased. However, owing to the high concentration of yeast cells the light may have hardly penetrated and its intensity attenuated drastically. As a result, the photosynthesis of algae was inhibited. The temperature 24 ℃ is sutible for the coculture of yeast and algae. The cells growth was stressed under an unsuitable cultivation. The biomass was decreased and the total lipids accumulation was increased about 33~37 %. Based on the light experiment, R. glutinis was promoted when S. obliquus photosynthesized and released O2 with the light. The growth of S. obliquus and R. glutinis were advanced except for the lipids accumulation with the light intensity raised. The higher growth rate of R. glutinis resulted in the shading effect to S. obliquus. Based on 5 L fermentor experiment, the effect of the coculture was advanced with 2 % CO2 added. Based on the 6 L air-lift bioreactor experiment, the biomass of 7.1 g/L was obtained at a coculture with 1.25 g/L KNO3 and 2 g/L YE added. However, the biomass of 6.1 g/L was obtained at a coculture with 0.5 g/L KNO3 added and without YE. It was shown that the shading effect is earthshaking compared to the nutrient in the medium.