Three-dimensional fibrillar networks reconstituted from collagen I are widely used as biomimetic scaffolds for in vitro and in vivo cell studies. Various physicochemical parameters of buffer conditions for in vitro fibril formation are well known, including pH-value, ion concentrations and temperature. However, there is a lack of a detailed understanding of reconstituting well-defined 3D network topologies, which is required to mimic specific properties of the native extracellular matrix. We screened a wide range of relevant physicochemical buffer conditions and characterized the topology of the reconstituted 3D networks in terms of mean pore size and fibril diameter. A congruent analysis of fibril formation kinetics by turbidimetry revealed the adjustment of the lateral growth phase of fibrils by buffer conditions to be key in the determination of pore size and fibril diameter of the networks. Although the kinetics of nucleation and linear growth phase were affected by buffer conditions as well, network topology was independent of those two growth phases. Overall, the results of our study provide necessary insights into how to engineer 3D collagen matrices with an independent control over topology parameters, in order to mimic in vivo tissues in in vitro experiments and tissue engineering applications.

Neurodegenerative disorders are characterised by the activation of brain-resident microglia cells and by the infiltration of peripheral T cells. However, their interplay in disease has not been clarified yet. It is difficult to investigate complex cellular dynamics in living animals, and simple two-dimensional (2D) cell culture models do not resemble the soft 3D structure of brain tissue. Therefore, we developed a biomimetic 3D in vitro culture system for co-cultivation of microglia and T cells. As the activation and/or migration of immune cells in the brain might be affected by components of the extracellular matrix, defined 3D fibrillar collagen I-based matrices were constructed and modified with hyaluronan and/or chondroitin sulphate, resembling aspects of brain extracellular matrix. Murine microglia and spleen-derived T cells were cultured alone or in co-culture on the constructed matrices. Microglia exhibited in vivo-like morphology and T cells showed enhanced survival when co-cultured with microglia or to a minor degree in the presence of glia-conditioned medium. The open and porous fibrillar structure of the matrix allowed for cell invasion and direct cell-cell interaction, with stronger invasion of T cells. Both cell types showed no dependence on the matrix modifications. Microglia could be activated on the matrices by lipopolysaccharide resulting in interleukin-6 and tumour necrosis factor-α secretion. The findings herein indicate that biomimetic 3D matrices allow for cocultivation and activation of primary microglia and T cells and provide useful tools to study their interaction in vitro.

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.