Criblage Combinatoire de Médicament en Microfluidique de Gouttes

Les puces microfluidiques sont de petites plateformes permettant de manipuler des fluides dans des canaux dont les dimensions sont à l'échelle submillimétrique. Ces dispositifs ont facilité la découverte de médicaments en augmentant le débit, le contrôle spatial de la diffusion des facteurs de signal, l'automatisation et la réduction du coût des expériences. La combinaison de médicaments est une stratégie alternative et prometteuse pour accélérer la découverte de médicaments, soit en combinant deux médicaments déjà approuvés, soit en combinant un candidat médicament avec un médicament approuvé. Le concept de combinaison d'antibiotiques s'aligne sur les preuves croissantes que les antibiotiques efficaces semblent interagir avec plus d'une cible. En général, la synergie n'est pas évidente à prévoir car elle résulte d'interactions complexes dans les voies cellulaires. L'exploration systématique de l'espace combinatoire pourrait être prolifique car des combinaisons synergiques non évidentes pourraient être identifiées.Dans cette thèse, nous avons proposé une nouvelle méthode basée sur des systèmes microfluidiques en réseau et des chambres d'hydrogel pour augmenter le débit des essais basés sur la diffusion tout en évitant un flux de travail complexe. Dans cette approche, les cellules restent dans la même position, et les réactifs sont amenés jusqu'à elles. Ainsi, chaque point reçoit un traitement connu, ce qui élimine la nécessité d'un marquage fluorescent ou d'un code-barres ADN. Deux gradients de concentration de petites molécules se chevauchant se forment dans chaque chambre cellulaire. Ainsi, contrairement aux plaques de microtitration où chaque puits contient une seule dose ou une seule combinaison de doses, notre puce microfluidique génère des combinaisons de doses multiples par chambre sans augmenter le travail de mélange des médicaments à diverses concentrations.J'ai commencé par décrire la conception de notre dispositif microfluidique qui distribue les composés médicamenteux dans les chambres, les dilue dans un hydrogel chargé de cellules et les mélange avec un autre composé. Pour fabriquer un tel dispositif, la principale difficulté est de fabriquer des membranes PDMS avec des trous traversants pour connecter une couche du dispositif microfluidique à la couche suivante. J'ai mis au point deux méthodes pour fabriquer des membranes en PDMS avec des micro-caractéristiques et des trous traversants accessibles.Pour montrer les performances de notre appareil, j'ai d'abord testé deux paires d'antibiotiques qui interagissent fortement : la combinaison sulfamonométhoxine × triméthoprime qui produit une interaction synergique et la combinaison FOS × nitrofurantoïne qui produit une interaction antagoniste. Nous avons calculé la surface des zones d'inhibition pour mesurer l'efficacité du traitement antibiotique. Nous avons ensuite testé la répétabilité des résultats en traitant toutes les chambres cellulaires avec la même paire d'antibiotiques à des concentrations identiques. Le coefficient de variabilité de l'aire des zones d'inhibition dans l'expérience de répétabilité était de 5,7 % pour le traitement avec une solution de 6,4 µg/ml de fosfomycine et de 7,6 % pour le traitement avec une solution de 20,48 µg/ml de nitrofurantoïne.En fin, j'ai expliqué l'itération à haut débit pour l'analyse de combinaisons de 12 antibiotiques. Nous avons démontré les interactions synergiques entre sulfamonométhoxine et triméthoprime et les interactions additives entre nitrofurantoïne et ampicilline. De plus, nous avons analysé statistiquement la répétabilité des traitements par médicament unique en calculant le coefficient de variation pour la taille des zones d'inhibition dans les hydrogels. Dans presque tous les cas, le coefficient de variation était inférieur à 10%, ce qui indique une bonne uniformité des résultats.J'ai consacré le dernier chapitre à discuter de cinq directions que nous pouvons poursuivre dans la suite de cette thèse.Microfluidic chips are small platforms to manipulate fluids in channels with dimensions at the submillimeter scale. These devices have facilitated drug discovery by increasing the throughput, spatial control on the diffusion of signal factors, automation, and reducing the cost of experiments. Drug combination therapy (DCT) is an alternative and promising strategy to accelerate drug discovery, either by combining two already approved drugs (repurposing by DCT) or by combining a drug candidate with an approved drug (boosting by DCT). The concept of antibiotic combination aligns with the increasing evidence that successful antibiotics seem to interact with more than one target. Considering the synergistic combination of β-lactam antibiotics and β-lactamase inhibitors as an exceptional case, the discovery of other combinations results from clinical experimentations. In general, synergy is non-obvious to predict as it arises from complex interactions in cellular pathways. The modern model of combinatorial discovery aims to systematically search for synergistic combinations instead of relying on the serendipitous discovery of synergy. Systematic exploration of combinatorial space could be prolific as non-obvious synergistic combinations might be identified.In this thesis, we proposed a novel method based on arrayed microfluidic systems and hydrogel chambers to increase the throughput of the diffusion-based assays while avoiding complex workflow. In this approach, the cells stay in the same position, and reagents are brought to them. Thus, each spot receives a known treatment, eliminating the need for fluorescent labeling or DNA barcoding. Two overlapping concentration gradients of small molecules will form in each cell chamber. Thus, in contrast to microtitration plates where each well contains a single dose or a single combination of doses, our microfluidic chip generates multiple-dose combinations per chamber without increasing the labor of mixing drugs at various concentrations.I began by describing the design of our microfluidic device which distributes the drug compounds to the chambers, dilutes them in a cell-laden hydrogel, and mixes them with another compound. To fabricate such a device, the main difficulty is fabricating PDMS membranes with through-layer holes to connect one layer of the microfluidic device to the next layer. I developed two methods for fabricating PDMS membranes with microfeatures and accessible through-layer holes.To show the performance of our device, first I tested two pairs of antibiotics that strongly interact: Sulfamonomethoxine × Trimethoprim combination that produces a synergistic interaction and Fosfomycin × Nitrofurantoin combination that produce an antagonistic interaction. We further tested the repeatability of the results by treating all the cell chambers with the same pair of antibiotics at identical concentrations. The coefficient of variability of the area of inhibition zones in the repeatability experiment was 5.7% for treatment with a 6.4 µg/ml solution of Fosfomycin and 7.6% for treatment with a 20.48 µg/ml solution of Nitrofurantoin, showing excellent reproducibility.Lastly, I explained the high-throughput iteration of the microdevice for analysis of combinations of 12 antibiotics. We demonstrated the synergistic interactions between Sulfamonomethoxine and Trimethoprim and additive interactions between Nitrofurantoin and Ampicillin. Further, we statistically analyzed the repeatability of single-drug treatments by calculating the coefficient of variation for the size of inhibition zones in the hydrogels. In almost all cases the CV was less than 10%, which indicates a good uniformity in the results.I dedicated the final chapter to discussing five directions we can pursue in the continuation of this thesis

Strategies for directing cells into building functional hearts and parts

This review presents the current state-of-the-art, emerging directions and future trends to direct cells for building functional heart parts.</p

Enhancing developmental rate and quality of mouse single blastomeres into blastocysts using a microplatform

The present work reports the beneficial effects of using a microplatform on the development of mouse single blastomeres (SBs) to the blastocyst stage. Development of blastocysts from SBs separated from two‐ and four‐cell stage embryos (two‐ and four‐cell SBs) can provide a valuable supply both for couples who use fertility‐assisted techniques and farm animals. As a step forward, we introduce three chips that provide the possibility of culturing SBs separately, in groups, and in the vicinity of the intact embryo (co‐culture), while each well of the chips is assigned to an isolated SB. Two‐ and four‐cell SBs co‐cultured with intact embryos showed 97.1% and 76.6% developmental rates and up to 34.1% and 49.1% growth relative to the microdroplet method (control). We examined the quality of developed blastocysts by assessing the total cell number, the number of inner cell mass (ICM) according to the octamer‐binding transcription factor 4 marker (OCT4), and trophectoderm (TE). Co‐culture of SBs with an intact embryo in a chip with nanoscale culture medium volume also increased the cell population of the developed embryo. The ICM:TE ratio, which is the most important blastocyst quality parameter, also indicated that developed two‐cell SBs have a higher degree of similarity to intact embryos despite fewer numbers of total cells

Oxygen-rich Environment Ameliorates Cell Therapy Outcomes of Cardiac Progenitor Cells for Myocardial Infarction

To some extent, cell therapy for myocardial infarction (MI) has supported the idea of cardiac repair; however, further optimizations are inevitable. Combined approaches that comprise suitable cell sources and supporting molecules considerably improved its effect. Here, we devised a strategy of simultaneous transplantation of human cardiac progenitor cells (CPCs) and an optimized oxygen generating microparticles (MPs) embedded in fibrin hydrogel, which was injected into a left anterior descending artery (LAD) ligating-based rat model of acute myocardial infarction (AMI). Functional parameters of the heart, particularly left ventricular systolic function, markedly improved and reached pre-AMI levels. This functional restoration was well correlated with substantially lower fibrotic tissue formation and greater vascular density in the infarct area. Our novel approach promoted CPCs retention and differentiation into cardiovascular lineages. We propose this novel co-transplantation strategy for more efficient cell therapy of AMI which may function by providing an oxygen-rich microenvironment, and thus regulate cell survival and differentiation

An integrated microfluidic device for stem cell differentiation based on cell-imprinted substrate designed for cartilage regeneration in a rabbit model

Separating cells from the body and cultivating them in vitro will alter the function of cells. Therefore, for optimal cell culture in the laboratory, conditions similar to those of their natural growth should be provided. In previous studies, it has been shown that the use of cellular shape at the culture surface can regulate cellular function. In this work, the efficiency of the imprinting method increased by using microfluidic chip design and fabrication. In this method, first, a cell-imprinted substrate of chondrocytes was made using a microfluidic chip. Afterwards, stem cells were cultured on a cell-imprinted substrate using a second microfluidic chip aligned with the substrate. Therefore, stem cells were precisely placed on the chondrocyte patterns on the substrate and their fibroblast-like morphology was changed to chondrocyte's spherical morphology after 14-days culture in the chip without using any chemical growth factor. After chondrogenic differentiation and in vitro assessments (real-time PCR and immunocytotoxicity), differentiated stem cells were transferred on a collagen-hyaluronic acid scaffold and transplanted in articular cartilage defect of the rabbit. After 6 months, the post-transplantation analysis showed that the articular cartilage defect had been successfully regenerated in differentiated stem cell groups in comparison with the controls. In conclusion, this study showed the potency of the imprinting method for inducing chondrogenicity in stem cells, which can be used in clinical trials due to the safety of the procedure