Progress in biomedicine requires the design of functional biomaterials, in particular, 3-dimensional (3D) scaffolds. Shear thinning, β-sheet based peptide hydrogels have attracted wide interest due to their potential use in tissue engineering and biomedical applications as 3D functional scaffolds. The emergence of carbon nanomaterials has also opened the door for the construction of increasingly functional hybrid hydrogels built from nanofibres and graphene-based materials using non-covalent physical interactions. The relationship between peptide molecular structure and the formed hydrogel is important for understanding the material response to shear. In particular, the physicochemical properties of peptide based biomaterials will affect the feasibility of injecting them during medical procedures. In the first part of this work, four peptides: FEFKFEFK (F8), FKFEFKFK (FK), KFEFKFEFK (KF8) and KFEFKFEFKK (KF8K) (F - phenylalanine, E - glutamic acid, K - lysine) were designed and used at identical charge to explore the effect of lysine rich β-sheet self-assembling sequences on the shear thinning behaviour and final properties of bulk hydrogels. By varying the peptide sequence design and concentration of the peptide, the tendency of the nanofibres formed to aggregate and the balance of nanofibre junction strength versus fibre cohesive strength could be explored. This allowed the existing theory of the shear thinning behaviour of this class of materials to be extended. The relationship between molecular structures of nanofibres forming the 3D network and the nano-filler is critical to understand in order to design tuneable and functional materials. In the next part of the work, three rationally designed β-sheet peptides, which form hydrogels: VEVKVEVK (V8), FEFKFEFK (F8) and FEFEFKFE (FE) (V - valine) and five graphene-based materials: graphene oxide (GO), reduced graphene oxide (rGO), three graphene-polymer hybrid flakes: GO with polydiallyldimethylammonium chloride (GO/PDADMAC), rGO with PDADMAC (rGO/PDADMAC) and rGO with polyvinylpyrrolidone (rGO/PVP) were used to form a selection of hybrid hydrogels. Graphene derivatives of the lateral flake sizes of 16.8 ± 10.1 µm were used. Various interactions between the graphene flakes and the peptides were observed that affected the overall mechanical properties of the hydrogels. Electrostatic interactions and pie-pie stacking, when phenylalanine residues are present, were shown to play a key role in determining the dispersion of graphene materials in the peptide hydrogels and stiffness of the hybrid materials. In particular, FE with reduced graphene oxide (rGO) and FE with rGO covered with polydiallyldimethylammonium chloride (PDADMAC) thin film formed double network-like hybrid hydrogels due to strong formation of peptide nanofibrillar bridges between adjacent rGO flakes. This corresponded to the 3- and 4-fold increase in the storage modulus (Gꞌ) of these hydrogels in comparison to controls. FE hydrogels with homogeneus dispersions of graphene oxide (GO) and reduced graphene oxide (rGO) are further shown to be suitable for 3D culture of human mesenchymal stem cells (hMSCs) with no cytotoxicity. These results focus attention on the importance of understanding interactions between the nano-filler and the nanofibrillar network in forming hybrid hydrogels with tuneable mechanical and biological properties, and demonstrates the possibility of using these materials as 3D cell culture scaffolds for biomedical purposes. Furthermore, graphene oxide (GO) itself is currently used in a number of processes of technological relevance such as wet spinning, injection moulding or inkjet printing to form graphene fibres, composites and printed conductors. Typically, such processes utilise well-aligned layered GO liquid crystal (LC) structures in aqueous dispersions. Flow and confinement encountered during processing affects the alignment and stability of this phase. In the final part of this work, the alignment of GOLCs of two lateral flake sizes (42.1 ± 29.4 µm and 15.5 ± 7.5 µm) were probed under a wide range of rotational shear flow conditions that overlap with the manufacturing processes defined by angular speeds from 0.08 to 8 rad.s-1 (and corresponding maximum shear rates from 0.1 s-1 to 100 s-1), in real-time, using shear induced polarized light imaging and small angle X-ray scattering, both coupled with an in-situ rheometer (Rheo-SIPLI and Rheo-SAXS, respectively). Under certain conditions, a unique pattern in Rheo-SIPLI: a Maltese cross combined with shear banding was observed. This phenomenon is unique to GO flakes of sufficiently large lateral size. The structure formed is attributed to a helical flow arising from a combination of shear flow and Taylor-vortex type flow, which is reinforced by a mathematical model. The orientations prescribed by this model are consistent with anomalous rheopecty oberved in Rheo-SIPLI and an anomolous scattering pattern in Rheo-SAXS. With the current trend towards producing ultra-large GO flakes, evidence that the flow behaviour changes from a Couette flow to a Taylor vortex flow was provided, which would lead to undesired, or alternatively, controllable alignment of GO flakes for a variety of applications, including aligned structures for biomedical purposes.

La détection de la protéine prion anormale dans les tissus lymphoïdes de patients britanniques suggère qu’après exposition à l’agent de la variante de la maladie de Creutzfeldt-Jakob (vMCJ) plus de 99% des contaminations pourraient demeurer cliniquement silencieuses. Ces données soulignent un risque de transmission secondaire par transfusion sanguine ce qui nous a conduit à une étude expérimentale. En parallèle des formes classiques de vMCJ, nos modèles murins et simiens de retransmission ont mis en evidence des phenotypes atypiques. Ces phénotypes échappent actuellement aux critères de diagnostic puisqu’aucune protéine prion anormale (PrPres) n’est détectée.Nos travaux ont eu pour but principal d’évaluer expérimentalement le risque sanguin au travers d’études de retransmission et de caractérisation de la replication des souches classiques et atypiques aux niveaux périphérique et central.Nous observons une très forte hétérogénéité dans la réplication de la PrP anormale dans les différents tissus lymphoïdes des macaques transfusés développant une vMCJ. Le niveau de contamination des tissus lymphoïdes apparait proportionnel à l’infectiosité sanguine de ces animaux et au risque de transmission de la maladie in vivo. Concernant les formes atypiques, la majorité des macaques transfusés n’ont pas de réplication dans les tissus lymphoïdes bien que ces phénotypes soient transmissibles expérimentalement à des modèles murins. Des transmissions à des souris immunodéficientes révèlent que les souches atypiques sont transmissibles par voie périphérique en l’absence d’un système immunitaire fonctionnel.Une alternative à l’expérimentation animale a été réalisée grâce aux « mini-brains » mimant la complexité du cerveau humain. Ces organoïdes cultivés en trois dimensions sont sensibles à au moins un isolat de prion associé aux formes sporadiques humaines. Les mini-brains pourraient ainsi constituer un nouvel outil d’étude des maladies à prions et permettre à termes la caractérisation des souches atypiques. / The detection of abnormal prion protein in the lymphoid tissues of UK patients suggests that after exposure to the agent of variant Creutzfeldt-Jakob disease (vCJD), more than 99% of contaminations may remain clinically silent. These data highlight a risk of secondary transmission through blood transfusion. In parallel to the classical vCJD forms, our experimental models in mice and macaques revealed another group which avoids the current diagnostic criteria, including the absence of abnormal prion protein (PrPres).The main goal of our work was to experimentally assess the risk of blood through retransmission studies and characterization of the abnormal replication of classical and atypical strains examined at peripheral and central levels.We observed a high heterogeneity of the distribution of the abnormal PrP in the lymphoid tissues of vCJD transfused macaques. The global level of contamination in lymphoid tissues seems proportional to the blood infectivity in these animals and to the risk of in vivo transmission of the disease. Regarding atypical forms, despite an absence of replication in lymphoid tissues, these phenotypes are experimentally transmissible. Transmissions to immunodeficient mice reveal that atypical strains are transmissible through peripheral routes in the absence of functional immune system.An alternative to animal testing has been achieved using to "mini-brains" mimicking the complexity of the human nervous system. These organoids cultured in three dimensions are sensitive to at least one prion isolate associated with human sporadic forms. Thus, mini-brains could constitute a new tool for studying prion diseases and improve the characterization of atypical strains.

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 géniques. / Skeletal 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.

Les matériaux à structure tridimensionnelle laissent entrevoir de nombreuses applications prometteuses dans le domaine de l'ingénierie tissulaire et de la médecine régénérative en fournissant un micro-environnement approprié pour l'incorporation de cellules ou de facteurs de croissance afin de régénérer des tissus ou organes endommagés. Dans ce contexte, une membrane a été élaborée à partir d'un mélange de poly (ε-caprolactone) PCL / chitosan CHT à partir d'une technique d'inversion de phase permettant un apport localisé de non solvent. La technique permet d'obtenir une double morphologie poreuse : (i) des macrovides en surface (gros pores) facilement accessibles pour l'invasion et la viabilité des cellules; (ii) un réseau macroporeux interconnecté (petits pores) pour transférer les nutriments, l'oxygène, le facteur de croissance à travers le matériau. Les propriétés physico-chimiques (taille des pores, chimie de surface et biodégradabilité) des matériaux ont été caractérisées. Il est montré comment il est possible d'ajuster les propriétés de la membrane en modifiant le rapport PCL / CHT. Des cultures de cellules souches mésenchymateuses humaines (CSMh) ont été réalisées sur la membrane. La viabilité et la prolifération cellulaires ont été étudiées par des essais de test au MTT et de taux d'absorption d'oxygène. Les expériences démontrent que la membrane est biocompatible et peut être colonisée par les cellules. La microscopie confocale montre que les cellules sont capables de pénétrer à l'intérieur des macrovides de la membrane. La prolifération cellulaire de CSM dans ce matériau pourrait être utile pour augmenter la longévité d'autres cellules primaires en modifiant les CSM pour produire des facteurs de croissance. Pour tester le comportement dynamique des cellules sur la membrane, un dispositif d'organe sur puce a été développé avec des cellules endothéliales ombilicales humaines ensemencées sur la membrane. Les résistances hydrauliques de la barrière cellulaire sur la membrane ont été quantifiées en temps réel pour une pression trans-endothéliale (PTE), 20 cm H2O à 37 ° C et avec des cellules vivantes après 1 jour et 3 jours après l'ensemencement. Les résultats suggèrent que ce type d'échafaudages polymères peut être utile à l'avenir comme patch in vivo pour réparer des vaisseaux endommagés. / Over the last decades, three-dimensional (3D) scaffolds are unfolding many promising applications in tissue engineering and regenerative medicine field by providing suitable microenvironment for the incorporation of cells or growth factors to regenerate damaged tissues or organs. The three-dimensional polymeric porous scaffolds with higher porosities having homogeneous interconnected pore network are highly useful for tissue engineering. In this context, a poly (ε- caprolactone) PCL/chitosan CHT blend membrane with a double porous morphology was developed by modified liquid induced phase inversion technique. The membrane shows: (i) surface macrovoids (big pores) which could be easily accessible for cells invasion and viability; (ii) interconnected microporous (small pores) network to transfer essential nutrients, oxygen, growth factors between the macrovoids and throughout the scaffolds. The physico-chemical properties (pore size, surface chemistry and biodegradability) of the materials have been characterized. This study shows how it is possible to tune the membrane properties by changing the PCL/CHT ratio. Human mesenchymal stem cell (hMSCs) culture was performed on the membranes and the cell viability and proliferation was investigated by MTT assay and oxygen uptake rate experiments. The experiments demonstrate that the membranes are biocompatible and can be colonized by the cells at micron scale. Confocal microscopy images show that the cells are able to adhere and penetrate inside the macrovoids of the membranes. Both cell proliferation and oxygen uptake increase with time especially on membranes with lower chitosan concentration. The presence of chitosan in the blend produces an increase of porosity that affect the entrapment of the cells inside the porous bulk of the membranes. Successful cellular proliferation of hMSCs could be useful to enhance longevity of other primary cells by production of corresponding growth factors. To test the dynamic behavior of cells on the membranes, an organ-on-chip (OOC) device has been developed with human umbilical endothelial cells (HUVECs) seeded on the membrane. The hydraulic resistance of the cellular barrier on the membrane has been quantified for real time trans-endothelial pressure (TEP) 20 cmH2O at 37 degree C and with living cells after 1 day and 3 day of post seeding. Results suggests this kind of polymeric scaffolds can be useful in future as an in vivo patch to repair disrupted vessels.

Au cours des 30 dernières années, d'importants progrès ont été faits dans le domaine de l'oncologie. Les cancers pédiatriques ont été les grands bénéficiaires des progrès des thérapies anticancéreuses et aujourd'hui, le cancer de l'enfant peut être soigné, dans les pays développés, dans 75 à 80% des cas. Cependant, ces thérapies sont connues pour leurs effets gamétotoxiques, et seulement 33 % des garçons qui ont survécu à leur cancer durant l'enfance produisent du sperme de bonne qualité une fois arrivé à l'âge adulte. Actuellement, la seule mesure de préservation envisageable pour ces enfants est de procéder à un prélèvement et à une cryoconservation de tissu testiculaire. Aujourd'hui, il est donc important de mettre au point un procédé capable de produire des spermatozoïdes à partir de tissus testiculaire dans le but de restaurer leur fertilité. Pendant plusieurs décennies, les biologistes de la reproduction ont essayé de développer une technologie pour accomplir in vitro la spermatogenèse chez les mammifères. Malgré des investissements importants dans la recherche, aucune méthode n'a permis de reproduire in vitro l'ensemble de ce processus chez l'homme. Dans cette étude, la société de biotechnologie Kallistem a développée, en collaboration avec des partenaires académiques incluant le laboratoire Ingénierie des Matériaux Polymères (projet ARTIS financé par la Canceropôle Lyon Auvergne Rhône-Alpes) un système de culture tridimensionnel constitué d'un hydrogel de chitosane capable de réaliser in vitro l'ensemble de la spermatogenèse chez différents mammifères incluant l'homme. Le système de culture 3D est un hydrogel physique de chitosane sous forme de tube obtenu après neutralisation d'une solution aqueuse de chitosane, sans aucun agent réticulant. Avantageusement, le tissu testiculaire est confiné dans la lumière du tube ce qui permet de conserver l'architecture 3D in vivo des tissus. L'influence de plusieurs paramètres structuraux du chitosane et de paramètres liés au procédé d'élaboration sur la microstructure, les propriétés mécaniques et de diffusion des hydrogels a été évaluée, dans le but d'optimiser la capacité du système de culture à assurer la survie et la différentiation cellulaire / During the past 30 years, huge progress has been performed in the field of oncology. In particular, pediatric cancers have been the beneficiaries and can now achieve cure rates of 75-80% in developed countries. However, cancer therapies are known for their gametotoxic effects and only 33% of male children who have survived cancer during childhood produce sperm of normality quality when they are adults. Currently, the only feasible conservation protocol for these boys is to make a collection and cryopreservation of their testicular tissue. There is thus a need to provide a process enabling to produce spermatozoa starting from testicular tissue in order to restore fertility. For several decades, reproductive biologists have been trying to develop a technology to achieve spermatogenesis in vitro in mammals. Despite sustained investment in research, no method has now reproduced in vitro this entire process in humans. In this work, Kallistem (Biotech Company) has developed, in collaboration with academic laboratories including “Polymer Materials Engineering” laboratory (project ARTIS financed by the Cancéropôle Lyon Auvergne Rhône-Alpes) a 3D culture system made of chitosan hydrogel enabling to make a complete spermatogenesis in vitro in several mammals including human. The 3D culture system is a tube of chitosan physical hydrogel obtained from neutralization of aqueous chitosan solution, without any external cross-linking agent. Advantageously, the testicular tissue is confined in the lumen of tube which enables to reproduce in vivo 3-dimensional architecture. The impact of several material and processing parameters on microstructure, mechanical and diffusion properties of resulting hydrogels was evaluated, in order to optimize the culturing and maturation ability of 3D culture system

No description available.

Bröstcancer är den vanligaste formen av cancer som drabbar kvinnor. Det är en heterogen och komplex sjukdom som består av flera undergrupper, var och en med distinkt morfologi och kliniska implikationer [1]. För att modellera och studera cellbiologi, vävnadsmorfologi, molekylära mekanismer och läkemedels effekter används cellkulturer [2]. Idag är tvådimensionella (2D) modeller fortfarande den mest använda metoden för att odla celler in vitro [3]. En nackdel med 2D-modeller är att mikromiljön i dessa modeller inte imiterar in vivo strukturen av tumörer och vävnader, då de saknar tre dimensionella (3D) cell-cell och cellextracellulär matrix (ECM) interaktioner [2]. På grund av nackdelarna med 2D-modeller, har 3D-modeller blivit mer intressanta som alternativ för att lösa behovet av en pålitlig preklinisk modell för läkemedelstestning och för studier av cancerbiologi. För att utveckla ett redskap som är relevant för cancerforskning etablerar professor My Hedhammars laboratorium en 3D-modell av bröstcancer. I en sådan ny modell används Biosilk som byggnadsställning för att odla odödliga cellinjer som är representativa för de tre huvudklasserna av bröstcancer (i.e. MCF-7 (luminal-lik), SKBR-3 (HER2-överuttryckt) och MDAMB- 231 (trippel-negativ)). Eftersom transkriptions signaturer kan användas för att klassificera och studera bröstcancer är det viktigt att undersöka om och hur tillväxt i 3D-Biosilk kan påverka genuttrycksprofiler. Hypotesen som testades i denna studie var om cellkulturer i 3DBiosilk kan ha signifikanta skillnader i uttryck av biomarkörer, relevanta för bröstcancerbiologi, vid jämförelse av samma cellinje kultiverad i 2D. För att testa detta utvärderades kvalitén och reproducerbarheten av 3D-Biosilk konstruktionen med hjälp av olika kvalitetstester. Strukturen granskades med brightfield mikroskopi, arean av konstruktionen mättes med ImageJ, infärgning med phalloidin bekräftade cellnärvaro och cellvidhäftning till modellen. Alamar blue utfördes för att bedöma den cellulära metaboliska aktiviteten i modellen. Förändringarna av målgenernas genuttryck undersöktes med kvantitativ omvänd transkription PCR (RT-qPCR) och detta påvisade en statistiskt signifikant skillnad i genuttrycket beroende på om cellerna odlats i 2D- eller 3D-Biosilk modeller. I cellinje MDA-MB-231 hittades tre gener, i cellinje SKBR-3 hittades två gener och i cellinje MCF-7 hittades fyra gener. Genuttrycket för en av dessa gener i cellinje MCF-7, som var kultiverad i 3D-Biosilk, var nedreglerad (i.e. ZO-1). Detta kunde valideras på proteinnivå med immunofluorescens. Sammanfattningsvis, celler odlade i 3D-Biosilk visar på en mer aggressiv fenotyp. / Breast cancer is the most common cancer among women. It is a heterogenous and complex disease composed of several subtypes, each with distinct morphological and clinical implications [1]. To model and study cell biology, tissue morphology, molecular mechanisms and drug actions, cell cultures are canonically used [2]. Today two-dimensional (2D) models are still widely the preferred method for culturing cells in vitro [3]. A drawback with 2D models is that the microenvironment in these models does not mimic the in vivo structure of tumors and tissues, lacking three-dimensional (3D) cell-cell and cell-extracellular matrix (ECM) interactions [2]. Due to the disadvantages of 2D models, 3D cultures have become an increasingly interesting alternative to solve the need for a reliable preclinical model for drug testing and the study of cancer biology. To develop a relevant tool for cancer research, the laboratory of professor My Hedhammar is currently establishing a 3D model of breast cancer. In such novel model, Biosilk is used as scaffold to grow immortalized cell lines representative of the three major classes of breast cancer (i.e. MCF-7 (luminal-like), SKBR-3 (HER2-overexpression) and MDA-MB-231 (triplenegative)). Since transcriptional signatures can be used to classify and study breast cancers, it is important to investigate if and how growth in 3D-Biosilk can impact gene expression profiles. The hypothesis tested in this study was that cells cultured in 3D-Biosilk have differences in expression of biomarkers relevant to breast cancer biology, when compared to the same cell lines cultured in 2D. To examine this, 3D-Biosilk models were created and evaluated to ensure their quality and reproducibility, for instance, the scaffold structure was monitored by brightfield microscopy, the construct’s area was measured with ImageJ, staining with phalloidin confirmed the presence of cells as well as their attachment to the construct, and Alamar blue was used to assess the cellular metabolic activity. Differences in gene expression of target genes were investigated using reverse transcription quantitative PCR (RTqPCR), which revealed statistically significant changes depending on whether the cells were cultivated in 2D or a 3D-Biosilk model. For cell line MDA-MB-231 three genes were found, for SKBR-3 two genes were found and for MCF-7 four genes were found. The expression of one gene which was found downregulated in MCF-7 cultured in 3D-Biosilk (i.e. ZO-1) was validated at protein level by immunofluorescence. In conclusion, cultivating cells in 3D-Biosilk indicates a more aggressive phenotype.

Le neuroblastome à haut risque est caractérisé par un faible taux de survie (~30%) et des récidives fréquentes, malgré les traitements multimodaux existants. Généralement, les études d’évaluation des médicaments utilisent la culture cellulaire en 2D, mais elle ne reflète pas la biologie tumorale du neuroblastome in situ, incluant les caractéristiques associées à l’hypoxie et la densité cellulaire. En comparaison aux patients, la culture cellulaire conventionnelle semble altérer le phénotype cellulaire du neuroblastome, le transcriptome et l’épigénome qui affecteront, à leur tour, les résultats des études pharmacologiques. Ainsi, un nouveau modèle de culture cellulaire en 3D a été développé avec plusieurs lignées cellulaires de neuroblastome afin d’atteindre une culture de sphéroïdes à long terme (un mois) avec un taux de viabilité convenable. L’hypothèse de recherche est que les changements épigénétiques et transcriptionnels seront induits par l’adaptation en 3D et vont s’amplifier dans le temps lors de la culture en 3D. Ces changements ont été mesurés en 3D dans le temps pour identifier le moment où l’épigénome des sphéroïdes ressemble le plus à celui des patient à haut risque. Le changement de l’expression des régulateurs épigénétiques survient 24 jours après la mise en sphéroïde, ce qui se traduit par une différence dans la sensibilité aux médicaments épigénétiques par rapport à la culture 2D. En conclusion, notre étude nous a permis de dériver des nouveaux modèles de neuroblastome qui sont plus représentatifs des patients d’un point de vue épigénétique et pharmacologique. / High-risk neuroblastoma is characterized by a low survival rate (~30%) and frequent recurrences, despite all the multimodal treatments available to date. Typically, drug evaluation studies use 2D cell culture which does not reflect well the tumor biology of neuroblastoma in situ, including features associated with hypoxia and cell density. Thus, compared to patients data, the conventional 2D cell culture seems to alter the neuroblastoma cell phenotype, transcriptome and epigenome which in turn will affect the results of pharmacological studies. A new 3D cell culture model was developed with several neuroblastoma cell lines to achieve long term spheroid culture (up to one month) with a suitable viability rate. We hypothesized that transcriptional and epigenetic changes will be induced by 3D adaptation and will amplify over time in the cells cultured in 3D. All these changes were measured by Western Blot in 2D, in short term 3D and in long term 3D to identify the timepoint when the epigenome of the spheroids most closely resembles that of the patient. We found that the occurrence of changes in epigenetic regulator expression occurs after 24 days of spheroid culture, resulting in a difference in a drug sensitivity compared to 2D culture. In summary, we developed new neuroblastoma models that are more representative of patient’s epigenome and pharmacological sensitivity.

To optimize neoadjuvant radiochemotherapy of pancreatic ductal adenocarcinoma (PDAC), the value of new irradiation modalities such as proton therapy needs to be investigated in relevant preclinical models. We studied individual treatment responses to RCT using patient-derived PDAC organoids (PDO). Four PDO lines were treated with gemcitabine, 5-fluorouracile (5FU), photon and proton irradiation and combined RCT. Therapy response was subsequently measured via viability assays. In addition, treatment-naive PDOs were characterized via whole exome sequencing and tumorigenicity was investigated in NMRI Foxn1nu/nu mice. We found a mutational pattern containing common mutations associated with PDAC within the PDOs. Although we could unravel potential complications of the viability assay for PDOs in radiobiology, distinct synergistic effects of gemcitabine and 5FU with proton irradiation were observed in two PDO lines that may lead to further mechanistical studies. We could demonstrate that PDOs are a powerful tool for translational proton radiation research.

Abstract  Lack of clinical relevance is assumed to be the main reason behind the high failure rate of medical drugs in the very initial phases of clinical trials. Clinical relevance is difficult to achieve with current tools as they lack the biological and physiological cues found in vivo. Microfluidics, the knowledge of fluid manipulation in small channels, has proven to be a promising science to bridge the gap between the current in vitro and the real in vivo features. In this thesis, a scaffold for the growth of spheroids inside a microfluidic device for potential drug screening was developed. Firstly, the surface of a microfluidic device was coated with the polymers cellulose nanofibrils, polyallylamine hydrochloride, and polyethyleneimine using the Layer-by-Layer technique to achieve an even surface coverage. Here, different chip designs, polymer concentrations, and pressure directions were tested. It was decided that using a negative pressure direction with a polymer concentration of 50 mg/L in a chip design with micropillars was optimal and these conditions were then used for testing the spheroid formation. Secondly, spheroids were grown inside the microfluidic channels using different coatings: the previously mentioned polymer buildup, one non-coated channel, and one coated with attachment factor proteins. These three surface conditions were compared and it was shown that the polymer-based surface cover was indeed superior as a scaffold as it encouraged and promoted cell growth in the spheroid formation of liver cancer cells from the HepG2 cell line. Further development of this cellulose nanofibrils-coated microfluidic device displays a promising future for functioning as an in vitro 3D cell culture model that better mimics the close-to-cell microenvironments by imitating cell proliferation, cell-to-cell, and cell-to-extracellular matrix interactions. / Sammanfattning Den främsta orsaken bakom den höga antal misslyckade kliniska läkemedelsprövningar i de initiala faserna antas bero på brist på klinisk relevans. Klinisk relevans är mycket svår att uppnå med dagens verktyg då de saknar de biologiska och fysiologiska förhållandena som återfinns in vivo. Mikrofluidik, kunskapen om vätskemanipulation i små kanaler har visat sig vara lovande vetenskap för att överbrygga klyftan mellan de nuvarande in vitro och de faktiska in vivo funktionerna. I detta arbete utvecklades en matris för sfäroider att växa på inuti en mikrofluidisk kanal för att potentiellt användas till läkemedelsscreening. Först användes Layer-by-Layer teknologi för att jämnt betäckta ytan inuti en mikrofluidisk kanal med polymererna cellulosananofibriller, polyallylamin hydroklorid samt polyetylenimin. Här testades olika designer på mikrofluidiska chip, polymerkoncentrationer samt tryckriktningar. Utifrån detta gick det att fastställa att negativt tryck med en polymerkoncentration på 50 mg/L i en chippdesign med mikropelare var optimal för en jämn ytbetäckning och dessa förhållanden användes sedan för att pröva sfäroidernas tillväxt. Härnäst testades därmed sfäroidernas tillväxt inuti mikrofluidiska kanaler under tre olika förhållanden: ett med polymerbetäckningen, ett utan betäckning och ett då ytan var täckt med proteiner med fästfaktorer. Dessa tre förhållanden jämfördes sedan med varandra och således gick det att konstatera att den polymerbaseradebetäckningen fungerade överlägset som matris för tillväxt av HepG2 lever cancer cell sfäroider eftersom den tycks främja dess tillväxt och bildning. Det pekar mot att ytterligare utveckling av denna cellulostäckta yta skulle innebära en lovande modell för in vitro 3D cellodling som bättre efterliknar den cellulära mikromiljön genom att imitera cellproliferation, interaktioner celler emellan samt mellan cell och extracellulär matrisen.