Developing an optimized growth environment for adipose-derived stems cells (ASCs) to obtain clinically useable cell quantities from relatively small tissue biopsies would significantly impact the field of tissue engineering. To date, ASCs have been differentiated into adipose, bone, cartilage, smooth muscle, endothelial, skeletal muscle, nervous, and cardiac tissue. Therefore, ASCs have potential for use in the treatment of a wide variety of clinical conditions ranging from myocardial infarction, to musculoskeletal disorders, and the repair of soft tissue defects. In this work, a custom-designed, 3-dimensional (3-D) scaffold-based perfusion bioreactor system was investigated in the culture of ASCs. Decellularized adipose tissue (DAT) was used to provide a 3-dimensional scaffold, as it possesses the native extracellular matrix (ECM) architecture and composition of human adipose tissue. The DAT had a permeability of 149 m2, based on a perfusion rate of 1.5 mL/min over a 200 mg DAT sample, and the culturing medium was evenly perfused throughout the DAT, thereby permitting possible cell growth within the central regions. Initial culturing studies of human ASCs on tissue culture polystyrene (TCPS) demonstrated that hypoxic (5% O2) conditions decreased the doubling time, and resulted in enhanced cell proliferation, as compared to normoxic (21% O2) conditions. The cell imaging and DNA quantification results showed that suspension seeding of the ASCs permitted cell attachment to the DAT scaffold, but did not support long-term ASC growth. In contrast, when the ASCs were seeded as multicellular aggregates, the cells attached and underwent measurable proliferation. The optimal seeding density observed was 1 x 106 ASCs/scaffold; or 50 aggregates (20,000 ASCs/aggregate) per scaffold. Based on the confocal imaging, the ASCs remained spherical in morphology during the entire culturing period. Moreover, results illustrated that the perfusion bioreactor provided an improved culturing environment for ASCs over traditional static culturing. Hypoxic (5% O2) conditions showed improved proliferation over normoxic (21% O2) conditions, within the bioreactor system. After a 14-day hypoxic culturing period in the perfusion bioreactor, the seeded ASCs retained the ability to undergo adipogenesis, as indicated by Glycerol-3-Phsophate Dehydrogenase (GPDH) enzymatic activity measurements, demonstrating the promise of this approach for soft tissue engineering applications / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-11-09 20:28:34.252

No description available.

Uma potencial alternativa para o transplante de tecidos é a engenharia de tecidos. Células-tronco mesenquimais e scaffolds eletrofiados são comumente utilizados nesta área devido à capacidade multipotente de diferenciação destas células e à rede de poros interconectados destas estruturas fibrosas. Além disso, bioreatores de perfusão podem ser utilizados para melhorar o transporte de nutrientes e reduzir o acúmulo de metabolitos tóxicos. Neste contexto, uma maneira de estudar e otimizar o sistema de cultivo é utilizar técnicas de modelagem para descrever interações ou processos individuais envolvidos no crescimento celular. Deste modo, o objetivo geral deste estudo é realizar o cultivo de células-tronco mesenquimais da polpa de dente decíduo (DPSCs) utilizando scaffolds tridimensionais eletrofiados de policaprolactona (PCL), biorreatores e técnicas de modelagem. Inicialmente foram testadas diferentes misturas de solventes (clorofórmio e metanol), a fim de produzir scaffolds com poros adequados ao cultivo tridimensional. Os diâmetros de fibra e de poro foram determinados por microscopia eletrônica de varredura (MEV). O crescimento e o metabolismo das células foram avaliados através da determinação da atividade metabólica e das concentrações de glicose e lactato do meio de cultivo, e a infiltração celular foi observada com a marcação do núcleo celular. Depois de estabelecidos os parâmetros de eletrofiação, o efeito da perfusão direta no desprendimento de DPSCs de scaffolds eletrofiados de PCL foi estudado. A atividade metabólica das células foi determinada para diferentes tempos de adesão, vazões e densidades de semeadura, e a tensão de cisalhamento na parede do poro foi calculada para cada vazão. A morfologia das células foi avaliada através de imagens de microscopia confocal e MEV. Paralelamente, foram realizadas simulações utilizando o software OpenFOAM para estudar como os parâmetros e variáveis de entrada (concentração inicial de glicose, porosidade e espessura do scaffold) afetam as saídas (fração volumétrica de células e concentração de substrato) de um modelo de proliferação celular que considera a difusão e o consumo de glicose. As contribuições do teor de oxigêno na cinética de crescimento de Contois e da variação da porosidade com o tempo devido à degradação do polímero também foram avaliadas. Inicialmente, foi observado que apenas um tamanho de poro maior que o diâmetro da célula permitiu a infiltração das células no scaffold. Então, observou-se que o aumento do tempo de adesão acarretou em maior espalhamento das células e, assim como a diminuição da densidade de semeadura e da tensão de cisalhamento, resultou em uma redução do desprendimento das células sob perfusão. Quanto ao modelo fenomenológico, observou-se maior sensibilidade à concentração inicial de glicose e à porosidade do scaffold, e aos parâmetros adimensionais relacionados à proliferação e morte celular e ao consumo de nutrientes. Além disso, o número inicial de células apresentou maior impacto no transporte de massa do que no crescimento celular. Neste estudo, foi possível obter scaffolds eletrofiados e conduções de cultivo dinâmico adequadas ao cultivo tridimensional de DPSCs, e elucidar os efeitos da limitação do transporte de massa e do oxigênio no crescimento celular, e da degradação do polímero no transporte de massa. / A potential alternative to tissue transplant is tissue engineering. Mesenchymal stem cells and electrospun scaffolds are commonly used in this field due to the multipotent differentiation capacity of these cells and the interconnected pore network of these fibrous structures. In addition, perfusion bioreactors can be used to enhance nutrient transport and reduce the accumulation of toxic metabolites. In this context, one way to study and optimize the culture system is to use modeling techniques to describe interactions or individual processes involved in cell growth. Thus, the objective of this study is to perform the three-dimensional culture of mesenchymal stem cells of dental pulp (DPSCs) using electrospun polycaprolactone (PCL) scaffolds, bioreactors and modeling techniques. Initially, different solvent mixtures (chloroform and methanol) were tested to produce scaffolds with pores suitable to three-dimensional culture. Fiber and pore diameter was determined using a scanning electron microscope. Cell growth and metabolism were evaluated through the metabolic activity and the culture medium concentration of glucose and lactate, and the cell infiltration was observed with cell nuclei staining. After the establishment of the elesctrospinning parameters, the effect of direct perfusion on DPSCs detachment from PCL electrospun scaffolds was investigated. The metabolic activity of the cells was determined for different adhesion times, flow rates and seeding densities and the pore wall shear stress was calculated for each flow rate. The cell morphology was evaluated through scanning electron and confocal microscopy imaging. In parallel, simulations with the software OpenFOAM were performed to study how parameters and inputs (initial glucose concentration, porosity and thickness of the scaffold) affect the outputs (cell volume fraction and substrate concentration) of a model of cell proliferation and glucose diffusion and consumption. The contribution of the oxygen in the Contois growth kinetics and the porosity variation with time due to polymer degradation was also evaluated. Initially, it was observed that only a pore size higher than the cell diameter allowed the infiltration of the cells through the scaffold. Then, it was observed that a higher adhesion time leaded to higher cell spreading in static conditions and, similar to smaller seeding densities and shear stresses, reduced cell detachment under perfusion. Regarding the phenomenological model, it was observed that the model is more responsive to the initial glucose concentration and scaffold porosity, and to the dimensionless parameters related to cell proliferation, death and nutrient uptake. Furthermore, the initial cell number had a more significant impact on mass transport than on cell growth. In this study, it was possible to obtain an electrospun scaffold and dynamic culture conditions suitable for the three-dimensional culture of DPSCs, and to elucidate the effects of transport limitations and of oxygen on cell growth, and of polymer degradation on mass transport were elucidated.

Tissue engineering strategies that combine porous biomaterial scaffolds with cells capable of osteogenesis or bioactive proteins have shown promise as effective bone graft substitutes. Attempts to culture bone tissue-engineering constructs thicker than 1mm in vitro often result in a shell of viable cells and mineralized matrix surrounding a necrotic core. To address this limitation, we developed a perfusion bioreactor system that improves mass transport throughout large cell-seeded constructs. Additionally, we established and validated 3-D computational methods to model flow and shear stresses within the microporosity of perfused constructs. Micro-CT scanning and analysis techniques were used to non-destructively monitor mineral development over time in culture. CFD modeling of axial perfusion through cylindrical scaffolds with a regular microarchitecture revealed a uniform flow field distributed throughout the scaffold. Perfusion resulted in a 140-fold increase in mineral deposition at the interior of 3 mm thick polymer scaffolds seeded with rat bone marrow stromal cells. The total detected mineral volume tripled as the construct length was increased from 3 to 9 mm. Increasing scaffold length to 9 mm did not affect the mineral volume fraction (MVF) within the full volume of each construct. Mineral volume, spatial distribution, density, particle size and particle number were then quantified on cell-seeded constructs in 5 different culture environments. The effect of time varying flow conditions was compared with continuous perfusion as well as two different control cell culture methods in an attempt to enhance mineralized matrix within the constructs. Intermittent elevated perfusion and dynamic culture in an orbital rocker plate produced the greatest amount of mineral within 9 mm long constructs compared to low continuous flow and high continuous flow cases. Together, these studies indicate that dynamic culture conditions enhance construct development with regards to cell viability, mineralized matrix deposition, growth rate, and distribution. Furthermore, these techniques provide a rational approach to selecting perfusion culture conditions that optimize the amount and distribution of mineralized matrix production. Finally, the established perfusion bioreactor, in combination with micro-CT analysis, provides a foundation for evaluating new scaffolds and cell types that may be useful for the development of effective bone graft substitutes.

Uma potencial alternativa para o transplante de tecidos é a engenharia de tecidos. Células-tronco mesenquimais e scaffolds eletrofiados são comumente utilizados nesta área devido à capacidade multipotente de diferenciação destas células e à rede de poros interconectados destas estruturas fibrosas. Além disso, bioreatores de perfusão podem ser utilizados para melhorar o transporte de nutrientes e reduzir o acúmulo de metabolitos tóxicos. Neste contexto, uma maneira de estudar e otimizar o sistema de cultivo é utilizar técnicas de modelagem para descrever interações ou processos individuais envolvidos no crescimento celular. Deste modo, o objetivo geral deste estudo é realizar o cultivo de células-tronco mesenquimais da polpa de dente decíduo (DPSCs) utilizando scaffolds tridimensionais eletrofiados de policaprolactona (PCL), biorreatores e técnicas de modelagem. Inicialmente foram testadas diferentes misturas de solventes (clorofórmio e metanol), a fim de produzir scaffolds com poros adequados ao cultivo tridimensional. Os diâmetros de fibra e de poro foram determinados por microscopia eletrônica de varredura (MEV). O crescimento e o metabolismo das células foram avaliados através da determinação da atividade metabólica e das concentrações de glicose e lactato do meio de cultivo, e a infiltração celular foi observada com a marcação do núcleo celular. Depois de estabelecidos os parâmetros de eletrofiação, o efeito da perfusão direta no desprendimento de DPSCs de scaffolds eletrofiados de PCL foi estudado. A atividade metabólica das células foi determinada para diferentes tempos de adesão, vazões e densidades de semeadura, e a tensão de cisalhamento na parede do poro foi calculada para cada vazão. A morfologia das células foi avaliada através de imagens de microscopia confocal e MEV. Paralelamente, foram realizadas simulações utilizando o software OpenFOAM para estudar como os parâmetros e variáveis de entrada (concentração inicial de glicose, porosidade e espessura do scaffold) afetam as saídas (fração volumétrica de células e concentração de substrato) de um modelo de proliferação celular que considera a difusão e o consumo de glicose. As contribuições do teor de oxigêno na cinética de crescimento de Contois e da variação da porosidade com o tempo devido à degradação do polímero também foram avaliadas. Inicialmente, foi observado que apenas um tamanho de poro maior que o diâmetro da célula permitiu a infiltração das células no scaffold. Então, observou-se que o aumento do tempo de adesão acarretou em maior espalhamento das células e, assim como a diminuição da densidade de semeadura e da tensão de cisalhamento, resultou em uma redução do desprendimento das células sob perfusão. Quanto ao modelo fenomenológico, observou-se maior sensibilidade à concentração inicial de glicose e à porosidade do scaffold, e aos parâmetros adimensionais relacionados à proliferação e morte celular e ao consumo de nutrientes. Além disso, o número inicial de células apresentou maior impacto no transporte de massa do que no crescimento celular. Neste estudo, foi possível obter scaffolds eletrofiados e conduções de cultivo dinâmico adequadas ao cultivo tridimensional de DPSCs, e elucidar os efeitos da limitação do transporte de massa e do oxigênio no crescimento celular, e da degradação do polímero no transporte de massa. / A potential alternative to tissue transplant is tissue engineering. Mesenchymal stem cells and electrospun scaffolds are commonly used in this field due to the multipotent differentiation capacity of these cells and the interconnected pore network of these fibrous structures. In addition, perfusion bioreactors can be used to enhance nutrient transport and reduce the accumulation of toxic metabolites. In this context, one way to study and optimize the culture system is to use modeling techniques to describe interactions or individual processes involved in cell growth. Thus, the objective of this study is to perform the three-dimensional culture of mesenchymal stem cells of dental pulp (DPSCs) using electrospun polycaprolactone (PCL) scaffolds, bioreactors and modeling techniques. Initially, different solvent mixtures (chloroform and methanol) were tested to produce scaffolds with pores suitable to three-dimensional culture. Fiber and pore diameter was determined using a scanning electron microscope. Cell growth and metabolism were evaluated through the metabolic activity and the culture medium concentration of glucose and lactate, and the cell infiltration was observed with cell nuclei staining. After the establishment of the elesctrospinning parameters, the effect of direct perfusion on DPSCs detachment from PCL electrospun scaffolds was investigated. The metabolic activity of the cells was determined for different adhesion times, flow rates and seeding densities and the pore wall shear stress was calculated for each flow rate. The cell morphology was evaluated through scanning electron and confocal microscopy imaging. In parallel, simulations with the software OpenFOAM were performed to study how parameters and inputs (initial glucose concentration, porosity and thickness of the scaffold) affect the outputs (cell volume fraction and substrate concentration) of a model of cell proliferation and glucose diffusion and consumption. The contribution of the oxygen in the Contois growth kinetics and the porosity variation with time due to polymer degradation was also evaluated. Initially, it was observed that only a pore size higher than the cell diameter allowed the infiltration of the cells through the scaffold. Then, it was observed that a higher adhesion time leaded to higher cell spreading in static conditions and, similar to smaller seeding densities and shear stresses, reduced cell detachment under perfusion. Regarding the phenomenological model, it was observed that the model is more responsive to the initial glucose concentration and scaffold porosity, and to the dimensionless parameters related to cell proliferation, death and nutrient uptake. Furthermore, the initial cell number had a more significant impact on mass transport than on cell growth. In this study, it was possible to obtain an electrospun scaffold and dynamic culture conditions suitable for the three-dimensional culture of DPSCs, and to elucidate the effects of transport limitations and of oxygen on cell growth, and of polymer degradation on mass transport were elucidated.

Uma potencial alternativa para o transplante de tecidos é a engenharia de tecidos. Células-tronco mesenquimais e scaffolds eletrofiados são comumente utilizados nesta área devido à capacidade multipotente de diferenciação destas células e à rede de poros interconectados destas estruturas fibrosas. Além disso, bioreatores de perfusão podem ser utilizados para melhorar o transporte de nutrientes e reduzir o acúmulo de metabolitos tóxicos. Neste contexto, uma maneira de estudar e otimizar o sistema de cultivo é utilizar técnicas de modelagem para descrever interações ou processos individuais envolvidos no crescimento celular. Deste modo, o objetivo geral deste estudo é realizar o cultivo de células-tronco mesenquimais da polpa de dente decíduo (DPSCs) utilizando scaffolds tridimensionais eletrofiados de policaprolactona (PCL), biorreatores e técnicas de modelagem. Inicialmente foram testadas diferentes misturas de solventes (clorofórmio e metanol), a fim de produzir scaffolds com poros adequados ao cultivo tridimensional. Os diâmetros de fibra e de poro foram determinados por microscopia eletrônica de varredura (MEV). O crescimento e o metabolismo das células foram avaliados através da determinação da atividade metabólica e das concentrações de glicose e lactato do meio de cultivo, e a infiltração celular foi observada com a marcação do núcleo celular. Depois de estabelecidos os parâmetros de eletrofiação, o efeito da perfusão direta no desprendimento de DPSCs de scaffolds eletrofiados de PCL foi estudado. A atividade metabólica das células foi determinada para diferentes tempos de adesão, vazões e densidades de semeadura, e a tensão de cisalhamento na parede do poro foi calculada para cada vazão. A morfologia das células foi avaliada através de imagens de microscopia confocal e MEV. Paralelamente, foram realizadas simulações utilizando o software OpenFOAM para estudar como os parâmetros e variáveis de entrada (concentração inicial de glicose, porosidade e espessura do scaffold) afetam as saídas (fração volumétrica de células e concentração de substrato) de um modelo de proliferação celular que considera a difusão e o consumo de glicose. As contribuições do teor de oxigêno na cinética de crescimento de Contois e da variação da porosidade com o tempo devido à degradação do polímero também foram avaliadas. Inicialmente, foi observado que apenas um tamanho de poro maior que o diâmetro da célula permitiu a infiltração das células no scaffold. Então, observou-se que o aumento do tempo de adesão acarretou em maior espalhamento das células e, assim como a diminuição da densidade de semeadura e da tensão de cisalhamento, resultou em uma redução do desprendimento das células sob perfusão. Quanto ao modelo fenomenológico, observou-se maior sensibilidade à concentração inicial de glicose e à porosidade do scaffold, e aos parâmetros adimensionais relacionados à proliferação e morte celular e ao consumo de nutrientes. Além disso, o número inicial de células apresentou maior impacto no transporte de massa do que no crescimento celular. Neste estudo, foi possível obter scaffolds eletrofiados e conduções de cultivo dinâmico adequadas ao cultivo tridimensional de DPSCs, e elucidar os efeitos da limitação do transporte de massa e do oxigênio no crescimento celular, e da degradação do polímero no transporte de massa. / A potential alternative to tissue transplant is tissue engineering. Mesenchymal stem cells and electrospun scaffolds are commonly used in this field due to the multipotent differentiation capacity of these cells and the interconnected pore network of these fibrous structures. In addition, perfusion bioreactors can be used to enhance nutrient transport and reduce the accumulation of toxic metabolites. In this context, one way to study and optimize the culture system is to use modeling techniques to describe interactions or individual processes involved in cell growth. Thus, the objective of this study is to perform the three-dimensional culture of mesenchymal stem cells of dental pulp (DPSCs) using electrospun polycaprolactone (PCL) scaffolds, bioreactors and modeling techniques. Initially, different solvent mixtures (chloroform and methanol) were tested to produce scaffolds with pores suitable to three-dimensional culture. Fiber and pore diameter was determined using a scanning electron microscope. Cell growth and metabolism were evaluated through the metabolic activity and the culture medium concentration of glucose and lactate, and the cell infiltration was observed with cell nuclei staining. After the establishment of the elesctrospinning parameters, the effect of direct perfusion on DPSCs detachment from PCL electrospun scaffolds was investigated. The metabolic activity of the cells was determined for different adhesion times, flow rates and seeding densities and the pore wall shear stress was calculated for each flow rate. The cell morphology was evaluated through scanning electron and confocal microscopy imaging. In parallel, simulations with the software OpenFOAM were performed to study how parameters and inputs (initial glucose concentration, porosity and thickness of the scaffold) affect the outputs (cell volume fraction and substrate concentration) of a model of cell proliferation and glucose diffusion and consumption. The contribution of the oxygen in the Contois growth kinetics and the porosity variation with time due to polymer degradation was also evaluated. Initially, it was observed that only a pore size higher than the cell diameter allowed the infiltration of the cells through the scaffold. Then, it was observed that a higher adhesion time leaded to higher cell spreading in static conditions and, similar to smaller seeding densities and shear stresses, reduced cell detachment under perfusion. Regarding the phenomenological model, it was observed that the model is more responsive to the initial glucose concentration and scaffold porosity, and to the dimensionless parameters related to cell proliferation, death and nutrient uptake. Furthermore, the initial cell number had a more significant impact on mass transport than on cell growth. In this study, it was possible to obtain an electrospun scaffold and dynamic culture conditions suitable for the three-dimensional culture of DPSCs, and to elucidate the effects of transport limitations and of oxygen on cell growth, and of polymer degradation on mass transport were elucidated.

L'ingénierie tissulaire représente une solution prometteuse pour la production de substituts osseux. L'utilisation de bioréacteurs à perfusion pour cultiver des cellules ostéo-compétentes sur des matrices poreuses, permet de résoudre les limitations dues au transfert de masse, et d'apporter des stimuli physiques améliorant la prolifération et la différenciation cellulaire. Malgré les récents et importants développements des bioréacteurs en ingénierie tissulaire, les mécanismes menant à la production de substituts osseux en bioréacteurs restent mal compris.Le but de cette thèse est d'améliorer la compréhension de l'influence des phénomènes de transport, sur la croissance cellulaire et tissulaire dans un bioréacteur à perfusion. Dans cet objectif, une approche combinant modélisation et expérimentation est proposée.Dans un premier temps, un cadre théorique rigoureux est développé afin d'étudier les propriétés de transport du bioréacteur. Etant donné la nature hiérarchique du système, l'aspect multi-échelle du problème doit être pris en compte. En se basant sur la méthode de prise de moyenne volumique avec fermeture, les processus de transport d'espèce et de quantité de mouvement sont homogénéisés à partir de l'échelle de la matrice extracellulaire, jusqu'à l'échelle du bioréacteur. Les propriétés effectives des différentes structures rencontrées sont évaluées, et l'influence des dépendances inter-échelles sont mises en valeur. Le model macroscopique obtenu inclus des termes non-conventionnels, dont les contributions sont évaluées pour les conditions de fonctionnement du bioréacteur.Dans un second temps, la prolifération cellulaire et la production de tissu sont étudiées d'un point de vue expérimentale et théorique. Premièrement, des cellules de type fibroblaste, sont cultivées jusqu'à trois semaines sur des billes de verre, dans un bioréacteur perfusé à 10mL/min. Un protocole combinant des techniques d'histologie et d'analyse d'image, permet de quantifier la croissance de cellules et de tissu en fonction du temps et de l'espace. Deuxièmement, une cinétique de production de tissu est introduite dans le modèle de transport multiéchelle développé plus tôt. Finalement, la résolution à l'échelle du bioréacteur permet de discuter les résultats expérimentaux et théoriques au regard des phénomènes de transport ayant lieux dans le bioréacteur à perfusion. / Tissue engineering represents a promising approach for the production of bone substitutes. The use of perfusion bioreactors for the culture of bone-forming cells on a three-dimensional porous scaffold material, resolves mass transport limitations and provides physical stimuli, increasing the overall proliferation and differentiation of cells. Despite the recent and important development of bioreactors for tissue engineering, the underlying mechanisms leading to the production of bone substitutes remain poorly understood. The aim of this thesis is to gain insight on the influence of transport phenomena, on cell and tissue growth within a perfusion bioreactor. To this purpose, a combined modeling and experimental approach is followed.To start with, a rigorous theoretical framework is developed in order to study the transport properties of the bioreactor. Given the hierarchical nature of the system, the multiscale aspect of the problem must be taken into account. Based on the volume averaging theory with closure, mass and momentum transport processes are upscaled from the extracellular matrix scale, to the bioreactor scale. The effective properties of the encountered structures are evaluated, and the influence of the interscale dependencies are emphasized. The resulting macroscopic model includes non-conventional terms, which contributions are evaluated in the case of the bioreactor culture conditions.Then, cell proliferation and tissue growth are studied both, from an experimental and modeling point of view. First, fibroblast cells are cultured on glass beads in a bioreactor, perfused with culture medium at 10mL/min, for up to three weeks. A protocol combining histological techniques and image analysis allows the quantification of cell and tissue growth as a function of space and time. Second, a theoretical tissue production kinetic is introduced in the multiscale transport model previously developed. Finally, the resolution at the bioreactor scale allows to discuss the theoretical and experimental results in regard to the transport phenomena taking place in the perfusion bioreactor.

La transplantation d’îlots pancréatiques offre un traitement potentielle pour le diabète de type 1 (T1DM). À ce jour, le succès mitigé de ce type de greffe est dû à plusieurs facteurs limitants comme le manque de revascularisation, la perte de la matrice extracellulaire (ECM) et le rejet par le système immunitaire du receveur. Dans les dernières années, l’utilisation de matrices tridimensionnelles (3D) et de bioréacteurs a amélioré le processus de transplantation et approfondi les connaissances sur le sujet. Le but de cette thèse est de mieux comprendre les effets des paramètres physiologiques (flux, concentration en oxygène dissous (D.O.) et pulsation) sur le tissu pancréatique dans un environnement 3D en utilisant un bioréacteur à perfusion. Le premier chapitre présente une revue de la littérature détaillant le pancréas, les maladies qui lui sont associées ainsi que les techniques permettant son étude in vitro et in vivo. L’utilisation de matrices 3D en recherche sur le diabète est discutée en profondeur tout en mettant l’emphase sur l’incorporation de molécules de la ECM. La revue souligne comment des matrices 3D testées en combinaison avec différents bioréacteurs ont permis de mieux comprendre et améliorer la culture de cellules pancréatiques. Une brève conclusion met en lumière les applications futures des bioréacteurs dans la recherche sur le diabète. La première étude de cette thèse traite de la culture de cellules de rat provenant d’insulinome (INS-1), encapsulées dans des matrices de fibrine en chambres de perfusion et cultivées dans un bioréacteur à perfusion. Un essai in situ de sécrétion d’insuline stimulée par le glucose fut développé pour comprendre les effets de la culture. Dans cette expérience, les effets bénéfiques des conditions contrôlées en bioréacteur à perfusion ont été démontrés et ont révélé une augmentation de l`indice de stimulation des cellules INS-1 avec le temps, une amélioration de la fonction GRIP, en plus d’une incidence moins élevée d’apoptose cellulaire en comparaison avec des témoins en culture statique, sans bioréacteur. Cette étude a été publiée dans la revue Biotechnology Progress. La deuxième étude décrit un design multifactoriel servant à l’identification des paramètres affectant des pancréas de rat dissociés mécaniquement, cultivés dans un bioréacteur à perfusion. Les effets uniques et combinés du flux, de la D.O. et de la pulsation ont été étudiés sur la culture de tissu pancréatique. Les conditions bénéfiques pour la culture en bioréacteur ont été identifiées. Le tissu pancréatique cultivé dans ces conditions bénéfiques a démontré une sécrétion d’insuline stimulée par le glucose, une plus grande activité métabolique, une coloration positive à l’insuline et au glucagon, des structures endothéliales multiples ainsi qu’un tissu plus intact en comparaison avec des cultures statiques cultivées en mode statique. Cette étude a été soumise à Biotechnology Progress. / Abstract : Transplantation of pancreatic islets offers a potential cure for type 1 diabetes mellitus (T1DM). To date, the success of such a graft has been mired by a number of limiting factors including lack of revascularisation, loss of native extracellular matrix (ECM), and graft rejection by the recipient’s immune system. In recent years, new ways to understand and improve this process have been explored using three-dimensional (3D) matrices and bioreactors. This thesis aims to further understand the important effect(s) physiological parameters (flow, dissolved oxygen concentration (D.O.) and pulsation) have on pancreatic tissue in a 3D environment using a perfusion bioreactor with defined geometries. The first chapter introduces a review of the literature detailing the native pancreas, its diseases, and how it is studied in vivo and in vitro. The use of 3D matrices in diabetes research is discussed with particular emphasis on the incorporation of ECM molecules. The review then highlights how 3D matrices have been used in combination with a host of different bioreactors to understand and improve pancreatic cell cultures. A brief conclusion about the future applications for the use of bioreactors in diabetes research is also discussed. The first experimental work comprises the culture of rat insulinoma cells (INS-1) encapsulated in fibrin matrices in perfusion chambers and cultured under perfusion bioreactor conditions. An in situ glucose-stimulated insulin secretion assay was then developed to monitor the culture over time. With this work, the beneficial effects of perfusion bioreactor conditions were shown and revealed increasing functionality (glucose-stimulated insulin secretion) of INS-1 cells over time, and a lower incidence of apoptosis when compared to static control cultures. This study was published in Biotechnology Progress. The second experimental work used a factorial design to identify process parameters affecting whole mechanically-disrupted rat pancreata in a perfusion bioreactor. Here, the singular and combinational effects of flow, dissolved oxygen concentration and pulsation were assessed on the outcome of pancreatic tissue. Beneficial bioreactor conditions were identified. Mechanically-disrupted rat pancreata cultured under these beneficial bioreactor conditions showed glucose-stimulated insulin secretion, higher metabolic activity, insulin- and glucagon-positive staining, extensive endothelial structures, and overall intact tissue when compared to static cultures. This study has been submitted to Biotechnology Progress.

L'ingénierie tissulaire intervient pour restaurer le tissu osseux. Parmi les traitements possibles, l'utilisation d'un bioréacteur à perfusion permet l'amplification in vitro de cellules souches ou osseuses prélevées chez le patient avant réimplantation. La contrainte de cisaillement générée par l'écoulement stimule mécaniquement les cellules et amplifie la production tissulaire. Cette technique souffre cependant de sa conception empirique et doit à présent être optimisée. L'objectif de cette thèse est l'étude et la modélisation de la croissance tissulaire et de la prolifération cellulaire à l'échelle du bioréacteur. En particulier, il s'agit de comprendre l'impact de l'écoulement sur la formation du tissu. Pour cela, une double approche de modélisation et d'expérimentation a été adoptée. Des expériences de culture cellulaire ont permis de mettre au jour la prolifération préférentielle des cellules près des parois du bioréacteur comme conséquence de l'hétérogénéité du support, et l'évolution de la morphologie du tissu. Un modèle prédisant le devenir des cellules ainsi que la croissance tissulaire à l'échelle du bioréacteur est proposé. L'aspect multi-échelles du problème est pris en considération et les procédures d'homogénéisation sont menées à bien grâce à la méthode de prise de moyenne volumique. / Bone tissue engineering aims at restoring bone tissues. Among the possible treatments, the use of a perfusion bioreactor allows the amplification in vitro of the patient bone or stem cells prior to implantation. The advantage of using such bioreactors is two-fold: in addition to greatly improving species transport, tissue production is enhanced. Although promising, this technique suffers from its empirical conception and now needs to be optimized. The purpose of this thesis is to study and model tissue growth and cell proliferation under a fluid flow of culture medium at the scale of the bioreactor. In particular, we wish to understand the impact of fluid flow on tissue formation. To this end, a double approach of experimentation and modeling has been adopted. Cell culture experiments in a perfusion bioreactor highlighted the preferential cell proliferation in the parietal region as a consequence of the heterogeneity of the scaffold, and the evolution of the tissue morphology. A model for predicting the cell's fate along with tissue growth at the scale of the bioreactor is proposed. The hierarchy of the system is considered and the upscaling procedures are carried out with the Volume Averaging Method.