In this work, a mathematical modelling approach is taken to improve and optimize the designs of bioartificial liver (BAL) systems. BALs are an alternative therapy for the extremely serious condition of liver failure where liver transplant is currently the only viable option. As yet, large-scale clinical trials have not been successful enough in order for BALs to gain regulatory approval. Through the work in this report, it is envisaged that BAL design can be improved to the point where they can gain clinical acceptanceOne of the main issues in BAL design is the provision of adequate oxygen to the cell mass. To this end, a mathematical model to describe oxygen mass transport is developed based on the principle of Krogh cylinders. The results of this model are subsequently interpreted and presented in Operating Region charts, an image of a parameter space that corresponds to viable BAL designs. These charts allow several important design trends to be identified, e.g. numerous short and thin hollow fibres are favourable over fewer thicker, longer fibres. In addition, it is shown that a physiologically relevant cell number of more than 10% of the native liver cell mass can be supported in these devices under the right conditions. Subsequently the concept of the Operating Region is expanded to include zonation, a metabolic phenomenon where local oxygen tension is a primary modulator of liver cell function. It is found that zonation profiles can be well controlled and under standard conditions a plasma flow rate of 185 ml/min to the BAL would distribute the three metabolic zones evenly. Finally, the principles of the Operating Region charts and zonation are applied to three existing commercial BAL designs; the HepaMate, BLSS and ELAD systems. In each case it could be seen that the default designs of each system did not present ideal environments for liver cells. Through consideration of zonation profiles, each device design and operating parameters could be optimized to produce in vivo-like environments. In the case of the ELAD, reducing the plasma flow rate from 500 to 90 ml/min resulted in a balanced zonation profile. Overall, the work in this report has developed and detailed a series of tools that will assist a BAL designer in making judicious choices over bioreactor design and operating parameters. As a result, it is hoped that BALs can take a step forward towards clinical practice and ultimately saving lives.

Currently, the only cure for liver failure is orthotopic liver transplantation. However, there are insufficient donor organs available to treat every patient on the transplant list and many die before they are able to receive a liver transplant. The bioartificial liver (BAL) device is a potential extracorporeal treatment strategy utilising hepatocytes or hepatocyte-like cells (HLCs) within a bioreactor to recapitulate normal liver function and therefore ‘bridge’ a patient with liver failure until they receive a transplant. The work in this thesis utilised tissue engineering methods to develop novel approaches to BAL device design through development and characterisation of a polymer membrane scaffold (“PX”) for hollow fibre bioreactor (HFB) culture and a HLC source generated from the transdifferentiation of pancreatic AR42J-B13 (B13) cells. A flat sheet membrane model was used for the development of asymmetrical, hydrophobic polystyrene (PS) phase inversion membranes. Oxygen plasma significantly increased PS membrane surface wettability through addition of oxygen functional groups to create an environment conducive for cell culture. The treated membrane was henceforth referred to as “PX”. The culture medium HepatoZYME+ was investigated for its ability to induce transdifferentiation of B13 cells to HLCs and maintain the hepatic phenotype. Overall, HepatoZYME+-cultured cells experienced viability loss. A diluted version, “50:50”, showed induction of the hepatic markers carbamoylphosphate synthetase-1 (CPS-1) and HNF4α, as well as a change towards a HLC morphology. When using 50:50 as a maintenance medium, transdifferentiated HLCs retained loss of pancreatic amylase and also induction of hepatic markers, with comparable serum albumin secretion to the established Dex + OSM treatment. However, culture viability in 50:50 was still compromised. Therefore, HepatoZYME+ based media were deemed unsuitable for induction and maintenance compared to Dex-based protocols. PX flat sheet membranes were able to support culture of B13 cells and also the human osteosarcoma cell line, MG63, demonstrating improved cell attachment over non-surface treated PS membranes. PX membranes supported transdifferentiation of B13 cells to HLCs, presenting with loss of pancreatic amylase, induction of the hepatic markers transferrin, GS and CPS-1 and serum albumin secretion. Furthermore, PX showed no change in mass or loss of culture surface area over 15 days in culture conditions. Together, the novel membrane material and the media formulation and feeding regime developed have strong potential to be translated to a HFB setting and guide future BAL device design.

Implanted tissue engineered substitutes constitute dynamic systems, with remodeling mediated by both the implanted cells and the host. Thus, there exists a significant need for methods to monitor the function and morphology of tissue engineered constructs. Noninvasive monitoring using 1H Nuclear Magnetic Resonance (NMR) spectroscopy and imaging can prove to be the solution to this problem. Spectroscopy allows for assessment of cellular function through the monitoring of inherent metabolic markers, such as total-choline, while high resolution imaging enables the evaluation of construct morphology and interfacial remodeling. We applied these 1H NMR methods to monitor betaTC3 mouse insulinoma cells within hydrogel-based materials as a model pancreatic tissue substitute. In vitro research established a strong correlation between total-choline, measured by 1H NMR spectroscopy, and viable betaTC3 cell number, measured by MTT. Extending these methods to in vivo monitoring, however, was met with additional challenges. First, the implanted cells needed to be contained within a planar construct above a threshold density to allow for adequate quantification of the total-choline peak. Secondly, cell-free buffer zones between the implanted cells and the host tissue needed to be incorporated to prevent host tissue signal contamination. Finally, quantitative techniques needed to be developed to accurately account for contaminating signal from diffusing molecules. To overcome these challenges, a disk-shaped agarose construct, initially containing a minimum of 4 million betaTC3 cells and coated with an outer layer of pure agarose, was fabricated. Mathematical simulations aided the implant design by characterizing diffusive transport of nutrients and metabolites into and out of the construct. In vivo 1H NMR studies of these constructs implanted in mice established a strong correlation between total-choline, measured noninvasively using 1H NMR spectroscopy, and viable cell number, measured invasively using MTT. This study establishes total-choline as a reliable marker for noninvasively quantifying dynamic changes in viable betaTC3 cell number in vivo. 1H NMR imaging was used to monitor the implants structural integrity over time, while also assessing the hosts fibrotic response. We expect these studies to establish quantitative criteria for the capabilities and limitations of NMR methodologies for monitoring encapsulated insulinomas, as well as other tissue implants.

The development of a bioartificial pancreas (BAP) has the potential to substantially improve the treatment of insulin-dependent diabetes. Composed of insulin-secreting cells encapsulated in a hydrogel material, a BAP may provide superior glycemic regulation compared with conventional exogenous insulin-delivery therapies. Towards this goal, β- cells or islets encapsulated in alginate microcapsules remain a promising approach. Due to the limited supply of human islets, alternative cell sources are under investigation for incorporation into a BAP, including porcine islets and β- cell lines. Several challenges remain to clinical implementation, including loss of islet or β- cell function and viability following transplantation and host response to the transplanted microcapsules. The objective of this work was to evaluate strategies to improve a BAP by supporting the function and survival of encapsulated islets and β -cells. Towards this goal, two areas were explored: 1) the provision of pro-survival and insulinotropic factors, namely, CXCL12 and GLP-1 (or a GLP-1 analog, Exendin-4), to encapsulated islets and β-cells and 2) modification of the alginate microcapsule to confer long-term resistance to host cell adhesion. To achieve the first objective, methods to deliver both pro-survival and insulinotropic factors to a BAP were developed and their effects on encapsulated β-cells and porcine islets were studied, both in vitro and in vivo. Results demonstrate that delivery of pro-survival and insulinotropic factors is a promising strategy to prolong the survival and function of a BAP. To reduce host cell adhesion to the microcapsule, we employed covalent conjugation of PEG to the surface of alginate-PLL capsules to replace the un-crosslinked layer of alginate used in traditional alginate-PLL-alginate (APA) microcapsules. Results demonstrate that while PEGylation of alginate-PLL microcapsules initially reduced host cell adhesion over 2 weeks in vivo compared with APA capsules, the PEG coating did not provide long-term protection over 3 months. Taken together, these studies represent a multipronged approach towards improving the duration of BAP function, with the ultimate goal of advancing this technology to the clinic.

La transplantation d’îlots pancréatiques est considérée comme une thérapie prometteuse quant au traitement du diabète de type 1. En revanche, l’utilisation de traitements immuno-suppresseurs ainsi que le manque de donneur sont un frein à l’expansion de cette thérapie à plus de patients diabétiques. Pour résoudre ces deux problèmes, la stratégie développée durant ces vingt dernières années est le pancréas bioartificiel. Il consiste en une immuno-isolation de la greffe dans une enveloppe artificielle, protégeant non seulement la greffe du système immunitaire, mais aussi le receveur de la greffe. Les îlots ou les cellules productrices d’insuline transplantée(e)s ne sont pas en contact avec le système immunitaire et aucune immunosuppression n’est nécessaire. L’objectif de ce travail était de déterminer les marqueurs de survie et de mort des îlots dans des conditions mimant celles du MAILPAN®, un pancréas bioartificiel développé par Defymed et d’étudier différentes molécules qui pourraient augmenter la survie des îlots. Nous avons démontré que cet environnement bioartificiel engendrait un confinement et de l’hypoxie créant un stress cellulaire et donc une perte précoce des îlots. Nous avons identifié plusieurs cibles pour améliorer la viabilité et la fonction comme par exemple les transporteurs d’oxygène ou des molécules anti-inflammatoires. Plus le nombre d’îlots dans le MAILPAN® était élevé, plus les effets délétères sur la survie des îlots étaient importants. En conséquence, nous avons testé différentes molécules impliquées dans les voies de l’hypoxie et de l’inflammation pour augmenter la survie et restaurer la fonction des îlots pancréatiques dans un environnement confiné et hypoxique (600IEQ/cm2). L’ajout d’HEMOXcell®, un nouveau transporteur d’oxygène et du peptide tat-CREB ont montré une restauration de la fonction des îlots ainsi qu’une diminution de l’hypoxie et de l’inflammation après 24h de culture. Ainsi ce travail a permis l’identification de nouveaux candidats pour l’élaboration d’un milieu spécialisé pour l’encapsulation de cellules. / Islet transplantation is considered as promising therapy for brittle type 1 diabetes. However, the use of immunosuppressive regimen and the lack of donor prevent the expansion of the therapy to other diabetic patients. In order to address these two issues, the strategy developed for the two last decades is the bioartificial pancreas. It consists in the immune-isolation of the graft in an artificial envelop, protecting at the same time the graft, from the immune-system, and the host, from the graft. In principle, the transplanted islets or surrogate insulin secreting cells are not in contact with the immune system and no immunosuppressive drugs are needed. The objective of this work was to identify the markers of islet death/survival mimicking MAILPAN® conditions, a bioartificial pancreas developed by Defymed and study different molecules which can improve islet survival.We demonstrated that bioartificial environment induced confinement and hypoxia which triggers cellular distress causing early islet loss. We identified several targets to improve viability and function such as oxygen carriers or anti-inflammatory drugs. The highest the number of islets in the MAILPAN® was, the most deleterious effects in islet survival and functionality were observed. As a consequence, we tested different molecules implicated in hypoxia and inflammation pathway to improve islet survival and restore islet functionality in a hypoxic and confined environment (600IEQ/cm²). The addition of HEMOXCell®, a novel oxygen carrier, and tat-CREB peptide have been shown to restore islets functionality and decrease hypoxia and inflammation levels after 24 hours in culture. Thus, these data provide new inputs for the design of a culture medium dedicated for cell encapsulation.

No description available.

Aujourd’hui, la transplantation est le seul traitement efficace proposé aux patients souffrant d’une insuffisance hépatique fulminante. La nécessité de disposer d’un système de suppléance hépatique transitoire apparaît donc indispensable. C’est dans cet axe que se sont développés les systèmes qualifiés de foies bio artificiels (BAL). Leur principale caractéristique est d’incorporer un bioréacteur hébergeant des cellules pouvant restaurer l’activité hépatiques dans son ensemble. A l’heure actuelle, les hépatocytes primaire humains (HEP) issus de foies de donneurs non transplantables sont considérées comme le meilleur choix. Cependant, leur utilisation reste limitée par leur faible disponibilité et la difficulté à les maintenir différenciés en culture in vitro. Pour remédier à ce dernier point, l’approche la plus prometteuse semble être une co-culture des hépatocytes avec les cellules non parenchymateuses afin de recréer un environnement proche des sinusoïdes hépatiques. Ce travail de thèse repose sur la mise en place d’une nouvelle approche de co-culture tridimensionnelle sous la forme de sphéroïdes, d’HEP primaires avec les principaux types de cellules non-parenchymateuses (les cellules de Kupffer, les cellules endothéliales et les cellules étoilées) selon des proportions spécifiques. Puis de leurs encapsulations dans des billes d’alginate et leurs cultures au sein d’un bioréacteur à lit fluidisé. Ce modèle s’est révélé pertinent et approprié à maintenir les fonctions hépatiques dans le temps. Bien que beaucoup d’optimisation reste à définir, ce travail exploratoire témoigne de l’intérêt de cette approche intéressante pour le progrès des systèmes BAL. / Liver shortage makes transplantation inapplicable to all acute liver failure patients. Bioartificial Iiver (BAL) devices represent a temporary solution for these patients which are thereby bridged tilt Iiver transplantation or regeneration BAL treatment offers blood purification and substitution of metabolic functions through the activity of hepatocytes (HEPs), which are integrated in the device within acclimating containers, so-called bioreactors. Primary human hepatocytes are the ideal cell type to use in BAL, but they are scarcely available and difficult to maintain in vitro. Co-culture of HEPs with supporting cells has been proposed as the most promising strategy for preserving HEP behaviors in in vitro conditions. In fact, assisting cells types hold their ability to influence functional responses of the HEPs by providing them with cues of the native organ.This PhD work proposed a novel approach of co-culture for the functional sustain and preservation of the HEPs in the environment of the fluidized bed bioreactor (designed in our Iaboratory). Definition of this model took inspiration from the cellular organization in the organ; therefore, it employed three major sinusoidal non-parenchymal cell populations (liver sinusoidal, Kupffer, and hepatic stellate cells) which, together with HEPs, were cultured with three-dimensional arrangement (spheroids) and according to specific proportions. The resulting model was characterized in terms of functional benefits for the HEPs, and then applied in the microenvironment of alginate beads, which provide cells with immunological and mechanical protection in the fluidized bed bioreactor. This spheroidal multi-cultured model revealed its potentiality in sustaining in vitro HEP behaviors over time. Although much remains to be refined, this model may represent an interesting approach for the progress of BAL

La nécessité de nouveaux modes de suppléance hépatique se fait clairement ressentir pour maintenir en vie les patients en attente d’une greffe. Des traitements, basés sur l’utilisation de cellules cultivées dans un environnement adéquat, pourraient même permettre le rétablissement de certains patients en insuffisance hépatique sévère et ainsi éviter la greffe et les traitements immunosuppresseurs associés. Une suppléance hépatique efficace pourrait aussi servir au rétablissement des patients récemment greffés ou ayant subi une hépatectomie. L’objectif de cette thèse a été de développer, créer et valider un nouveau foie bioartificiel avec une prise en compte des contraintes réglementaires des médicaments combinés de thérapie innovante. Ce BAL doit pouvoir inclure des bioréacteurs à lit fluidisé perfusés par du plasma et contenant des sphéroïdes d’hépatocytes encapsulés. Des microparticules de verre, ajoutés aux billes d’alginate pour les alourdir, permettent d’obtenir une fluidisation optimale dans du plasma pathologique sans que des effets délétères ne soient observables ni pour les cellules ni pour les propriétés mécaniques des billes. Une méthode de culture cellulaire utilisant un revêtement anti-adhérent sur des boites de Petri en verre permet de produire un nombre important de sphéroïdes viables in vitro. Ces sphéroïdes encapsulés peuvent être maintenus vivants et métaboliquement actifs dans un bioréacteur à lit fluidisé pendant au minimum 4 jours.Pour assurer l’efficacité du BAL et la sécurité des patients, une circulation extracorporelle complexe a été mise au point pour être compatible avec une machine d’épuration extracorporelle commerciale, la Prismaflex® de la société Gambro, déjà utilisée en soins intensifs. Une étude préclinique sur un modèle ovin a montré que le traitement était bien toléré en ce qui concerne les aspects hémodynamiques. La prochaine étape concerne la mise en place d’une étude dans un modèle porcin d’insuffisance hépatique, avant de pouvoir procéder aux premiers essais cliniques. / The need for new liver support devices is clearly felt to allow keeping alive patients waiting for a transplant. Treatments, based on the use of cells cultured in an adequate environment, may even allow the recovery of some patients suffering from acute liver failure and avoid graft and associated immunosuppressive therapies. A hepatic substitution could also be used to reestablish patients recently transplanted or who had underwent an hepatectomy.. The objective of the thesis was to design, create and validate of a new bioartificial liver with consideration for the regulatory requirements of the Advanced Therapies Medical Product (ATMP). This device has to include fluidized bed bioreactors perfused with plasma and hosting alginate-encapsulated hepatocytes spheroids. Microparticles of glass have been added to weight down alginate beads in order to have an optimal fluidization in pathological plasma without negative effects neither on cells metabolism nor on mechanical properties of the beads. A cellular culture method using non adhesive coating in Petri dish led to the production of a large amount of viable spheroids in vitro. These encapsulated spheroids can be kept alive and metabolically active in a fluidized bed bioreactor during a minimum of four days. To ensure the efficacy of the BAL and the safety of patients, a complex extracorporeal circulation was designed to be compatible with a commercial medical device, the Prismaflex® monitor of the company Gambro, already used in intensive care units. A preclinical study on sheeps has shown that the treatment was well tolerated in terms of hemodynamics considerations. The next step is the establishment of a study in a porcine model of liver failure, before we can proceed to the first clinical trial.

No description available.