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Organ-on-Chip (OoC) systems culture human tissues in a controllable environment under microfluidic perfusion and enable a precise recapitulation of human physiology. Although recent studies demonstrate the potential of OoCs as alternative to traditional cell assays and animal models in drug development as well as personalized medicine, unmet challenges in device fabrication, parallelization and operation hinder their widespread application. In order to overcome these obstacles, this thesis focuses on the development of the Organ-on-a-Disc technology for the scalable generation and cultivation of microphysiological tissues. Organ-Discs are fabricated using precise, rapid and scalable microfabrication techniques. They enable the pump- and tubing-free perfusion as well as the parallelized generation and culture of tailorable and functional microtissues using rotation-based operations. The Organ-Disc setup is suitable for versatile tissue readouts, treatments and even whole blood perfusion with minimal handling and equipment requirements. Overall, the Organ-Disc creates a scalable and userfriendly platform technology for microphysiological tissue models and paves the way for their transition towards high-throughput systems.:Abbreviations Symbols 1 Introduction 2 Background 2.1 Fluid Dynamics 2.1.1 Flow Equations 2.1.2 Hydraulic Resistance 2.1.3 Wall Shear Stress 2.1.4 Centrifugal Microfluidics 2.2 Microfluidic Chip Fabrication 2.2.1 Chip Materials 2.2.2 Microstructuring 2.2.3 Bonding 3 State of the Art 3.1 Cell Culture Systems 3.2 3D Tissue Generation in Microfluidic Systems 3.3 Organ-on-Chip 3.4 Scale-up of Organ-on-Chip Systems 3.4.1 Scalable Fabrication Technologies 3.4.2 Parallelization Approaches 3.4.3 Integrated Fluid Actuation 3.5 Centrifugal Microfluidics 4 Objectives 5 Materials and Methods 5.1 Organ-Disc Fabrication 5.1.1 Materials 5.1.2 2D Structuring 5.1.3 Hot Embossing Stamp Fabrication TPE Hot Embossing 5.1.4 Bonding Solvent Vapor Bonding Thermal Fusion Bonding TPE Bonding 5.1.5 Characterization Methods Structure Sizes Bonding Strength Optical Properties 5.2 Organ-Disc Spinner 5.2.1 Centrifugal Loading Setup 5.2.2 Centrifugal Perfusion Setup 5.2.3 Peristaltic Pumping Setup 5.3 Organ-Disc Perfusion 5.3.1 Centrifugal Perfusion 5.3.2 Peristaltic Perfusion 5.4 Preparatory Cell Culture 5.5 Organ-Disc Cell Loading 5.5.1 Centrifugal Cell Loading 5.5.2 Endothelial-lining 5.6 Organ-Disc Cell Culture 5.6.1 Staining and Imaging Live Cell Labeling Live/Dead Staining CD106 Staining CD41 Staining Fixation, Permeabilization and Blocking Actin/Nuclei Staining CD31/Nuclei Staining 5.6.2 Media Analysis 5.6.3 Endothelial Cell Activation 5.6.4 Whole Blood Perfusion 5.7 Data Presentation and Statistics 6 Concept and Design 6.1 Organ-Disc Technology 6.2 Organ-Disc Design 6.3 Centrifugal Cell Loading 6.4 Endothelial Cell Lining 6.5 Centrifugal Perfusion 6.6 Peristaltic Perfusion 7 Building Blocks 7.1 Microfabrication Technology 7.1.1 Structuring 2D Structuring Hot Embossing 7.1.2 Bonding Solvent Vapor Bonding Thermal Fusion Bonding TPE Bonding 7.2 Organ-Disc Spinner 8 Perfusion 8.1 Centrifugal Pumping 8.2 Peristaltic Pumping 9 Tissue Generation and Culture 9.1 3D Tissue Generation 9.2 Stratified Tissue Construction 9.3 Generation of Endothelial-lined Channels 9.4 Perfusion of Endothelial-lined Channels 9.4.1 Media Monitoring Evaporation Cell Metabolism 9.4.2 Inflammatory Cell Stimulation 9.4.3 Whole Blood Perfusion 10 Discussion 10.1 Organ-Disc Technology 10.2 Scalable, Precise and Robust Organ-Disc Fabrication 10.2.1 Fabrication of Thermoplastic Organ-Discs 10.2.2 Fabrication of TPE Modules 10.2.3 Integration of TPE Modules to Organ-Discs 10.3 Tunable, Pump- and Tubing-free Perfusion 10.4 On-Disc Tissue Culture 10.4.1 3D Tissues 10.4.2 Blood Vessel-like Structures 10.4.3 Tissue Characterization and Treatment 10.5 On-Disc Blood Perfusion 11 Summary and Conclusion 12 References 13 Appendix / In Organ-on-Chip (OoC)-Systemen werden menschliche Gewebe mittels mikrofluidischer Versorgung in einer kontrollierten Umgebung kultiviert und so die Physiologie des Menschen nachgebildet. Obwohl aktuelle Studien zeigen, dass dieser Ansatz Alternativen zu herkömmlichen Zellbasierten Tests und Tiermodellen in der Arzneimittelentwicklung und der personalisierten Medizin bietet, stehen einer breiteren Anwendung Hürden im Bereich der Herstellung, Parallelisierung und Handhabung im Weg. Deshalb ist das Ziel dieser Arbeit die Entwicklung der Organ-on-a-Disc-Technologie, die eine skalierbare Erzeugung und Kultur von mikrophysiologischen Geweben ermöglicht. Für die Herstellung von der Organ-Disc kommen präzise, schnelle und skalierbare Mikrofabrikationsmethoden zum Einsatz. Die Organ-Disc schafft die Basis für die parallelisierte Erzeugung und Kultur von maßgeschneiderten und funktionellen Mikrogeweben, sowie deren Versorgung durch rotationsbasierte Prozesse und ohne zur Hilfenahme von Pumpen oder Schläuchen. Die Organ-Disc eignet sich für unterschiedliche Charakterisierungsmethoden sowie der Gewebestimulation und sogar der Vollblutperfusion mit minimalem Aufwand und Equipment. Insgesamt stellt die Organ-Disc eine skalierbare und benutzerfreundliche Plattformtechnologie für mikrophysiologische Modelle dar und bereitet den Weg für Hochdurchsatzanwendungen.:Abbreviations Symbols 1 Introduction 2 Background 2.1 Fluid Dynamics 2.1.1 Flow Equations 2.1.2 Hydraulic Resistance 2.1.3 Wall Shear Stress 2.1.4 Centrifugal Microfluidics 2.2 Microfluidic Chip Fabrication 2.2.1 Chip Materials 2.2.2 Microstructuring 2.2.3 Bonding 3 State of the Art 3.1 Cell Culture Systems 3.2 3D Tissue Generation in Microfluidic Systems 3.3 Organ-on-Chip 3.4 Scale-up of Organ-on-Chip Systems 3.4.1 Scalable Fabrication Technologies 3.4.2 Parallelization Approaches 3.4.3 Integrated Fluid Actuation 3.5 Centrifugal Microfluidics 4 Objectives 5 Materials and Methods 5.1 Organ-Disc Fabrication 5.1.1 Materials 5.1.2 2D Structuring 5.1.3 Hot Embossing Stamp Fabrication TPE Hot Embossing 5.1.4 Bonding Solvent Vapor Bonding Thermal Fusion Bonding TPE Bonding 5.1.5 Characterization Methods Structure Sizes Bonding Strength Optical Properties 5.2 Organ-Disc Spinner 5.2.1 Centrifugal Loading Setup 5.2.2 Centrifugal Perfusion Setup 5.2.3 Peristaltic Pumping Setup 5.3 Organ-Disc Perfusion 5.3.1 Centrifugal Perfusion 5.3.2 Peristaltic Perfusion 5.4 Preparatory Cell Culture 5.5 Organ-Disc Cell Loading 5.5.1 Centrifugal Cell Loading 5.5.2 Endothelial-lining 5.6 Organ-Disc Cell Culture 5.6.1 Staining and Imaging Live Cell Labeling Live/Dead Staining CD106 Staining CD41 Staining Fixation, Permeabilization and Blocking Actin/Nuclei Staining CD31/Nuclei Staining 5.6.2 Media Analysis 5.6.3 Endothelial Cell Activation 5.6.4 Whole Blood Perfusion 5.7 Data Presentation and Statistics 6 Concept and Design 6.1 Organ-Disc Technology 6.2 Organ-Disc Design 6.3 Centrifugal Cell Loading 6.4 Endothelial Cell Lining 6.5 Centrifugal Perfusion 6.6 Peristaltic Perfusion 7 Building Blocks 7.1 Microfabrication Technology 7.1.1 Structuring 2D Structuring Hot Embossing 7.1.2 Bonding Solvent Vapor Bonding Thermal Fusion Bonding TPE Bonding 7.2 Organ-Disc Spinner 8 Perfusion 8.1 Centrifugal Pumping 8.2 Peristaltic Pumping 9 Tissue Generation and Culture 9.1 3D Tissue Generation 9.2 Stratified Tissue Construction 9.3 Generation of Endothelial-lined Channels 9.4 Perfusion of Endothelial-lined Channels 9.4.1 Media Monitoring Evaporation Cell Metabolism 9.4.2 Inflammatory Cell Stimulation 9.4.3 Whole Blood Perfusion 10 Discussion 10.1 Organ-Disc Technology 10.2 Scalable, Precise and Robust Organ-Disc Fabrication 10.2.1 Fabrication of Thermoplastic Organ-Discs 10.2.2 Fabrication of TPE Modules 10.2.3 Integration of TPE Modules to Organ-Discs 10.3 Tunable, Pump- and Tubing-free Perfusion 10.4 On-Disc Tissue Culture 10.4.1 3D Tissues 10.4.2 Blood Vessel-like Structures 10.4.3 Tissue Characterization and Treatment 10.5 On-Disc Blood Perfusion 11 Summary and Conclusion 12 References 13 Appendix

<p>Microdroplets have large and varied areas of application ranging from document printing to complex lab-on-chip devices. Lab-on-chip systems often require precise volume control as well as high throughput operations. Microdroplets fulfill these requirements and have become a staple in these devices. The work presented in this thesis involves the design and characterization of two individual devices capable of droplet generation utilizing flow focusing and electrogeneration methods.</p> <p>The first design involved the generation of gel microdroplets utilizing the flow focusing technique. This device proved to be robust and reliable producing large volumes of uniformly mixed droplets. Long term operation of this device was analyzed and determined to be a feasible route for the manufacture of large quantities of droplets. The device was operated for over 30 hours creating gel droplets ranging from 40-200 μm in diameter with acceptable polydispersities for use in drug release studies.</p> <p>The second device involved the design and characterization of a system for the electrogeneration of microdroplets. This novel device involved the injection of droplets via high voltage and high frequency signals into a cross-flow of oil. The droplet generation was characterized and different droplet generation modes were observed. With the careful selection of parameters ideal conditions were obtained to generate monodisperse droplets of sizes ranging from under 5 to over 100 μm in a highly repeatable manner.</p> <p>To conclude, two separate microfluidic droplet generation devices operating in distinct modes were designed and analyzed. These devices are robust, reliable, and flexible with some applications being tested.</p> / Master of Applied Science (MASc)

<p>A microfluidic device capable of drug delivery to multiple wells in a concentration gradient was designed for automated high content and high throughput screening. The design was proposed to utilize a nanoporous polycarbonate membrane to spatially and temporally control drug dosage from the microchannels below to the wells above. Microchannels were to hold to the drugs or reagents, while wells were to culture cells. An array of 16 wells was to fit in the equivalent area of a single well of a 96 well plate. Two simpler devices were created to validate electrokinetic drug delivery to a single well and to characterize cell proliferation and viability in micro-wells. The first device tested drug delivery to a single well with methylene blue dye at applied voltages of 100V, 125V, and 150V. It was validated that the dosage of dye could be controlled by increasing the voltage and by increasing the duration the voltage was applied. The second devices were a series of 9-well arrays, each testing a different diameter (1.2 mm – 0.35 mm). These devices were cultured with MCF-7 breast cancer cells over 5 days. At the end of the 5 day study, all diameters except for 0.5 mm and 0.35 mm measured a cell viability of 99% and exhibited cell growth patterns similar to coverslip glass controls. The proposed integrated cell culture and drug delivery device could have application towards early stage drug discovery and could have compatibility with lab equipment originally designed for well plates.</p> / Master of Applied Science (MASc)

Infertility is a global challenge, often remedied with In vitro fertilization (IVF) and Intra cytoplasmic sperm injection (ICSI). Sperm quality is crucial, prompting ICSI when compromised. Routine sperm preparation via gradient centrifugation raises concerns about sperm stress and DNA fragmentation. Zymot, a new device that utilizes microfluidic technology, emerges as a promising alternative. It minimizes sperm stress and DNA damage, potentially enhancing fertilization rates and embryo quality. The aim of this study was to compare the outcome of ICSI samples treated with Zymot against gradient centrifuged samples. Focus was on fertilization rates, embryo quality, and pregnancy outcomes.  The results of 104 Zymot treated samples from men with compromised sperm quality were compared with 144 gradient centrifugations retrospective. Results revealed a significant difference between methods in the number of pronulear (PN), specifically in the Good Quality Embryo (GQE) where 62% with Zymot were 2PN compared to 59% with gradient (p=0.017). No significant difference in pregnancy rates or embryo utilization rate were observed. A tendency towards higher proportion (54.6%) of Zymot-treated embryos were cryopreserved compared to gradient (49.4%, p=0,27). In conclusion, a significant difference between methods in the GQE proportion of 2PN embryos favored Zymot. Closer examination revealed a higher proportion of embryos cryopreserved with Zymot, suggesting a potential for increased treatment success in future cycles. Zymot, requiring less time, yielded equivalent results to gradient centrifugation, with higher GQE proportions and more embryos cryopreserved. This merits consideration as a high-quality alternative to sperm preparation for ICSI in cases of poor sperm quality.

La leucémie aigüe lymphoblastique de type B (B-LAL) est la principale cause de mortalité par maladie chez les enfants. Bien que la chimiothérapie moderne puisse guérir la majorité des patients, environ 20 % d'entre eux présentent une réponse inadéquate ou vont rechuter. La stratification des patients en sous-types moléculaires distincts grâce à la cytogénétique, à la biologie moléculaire et au profilage transcriptomique est essentielle pour adapter les thérapies en fonction des risques. Le temps nécessaire à l'analyse traditionnelle des échantillons de tumeurs empêche une intervention rapide au début du traitement. Comme alternative aux biopsies tissulaires conventionnelles, nous proposons que les vésicules extracellulaires (VE), particulièrement les exosomes présents dans le sang périphérique, sont des transporteurs potentiels de biomarqueurs tels que l’ARN. Grâce à l'analyse d'une cohorte de patients représentant deux sous-types moléculaires distincts de la B-LAL, des transcrits d'ARN encapsulés dans les exosomes ont été identifiés comme biomarqueurs non invasifs prometteurs. Ces résultats plaident en faveur d'une détection précoce et d'un suivi continu de la B-LAL de l’enfant à l'aide de biopsies liquides basées sur les exosomes. Néanmoins, le processus de purification des exosomes, long et complexe, nécessite une normalisation et une amélioration afin d'établir une crédibilité dans le suivi clinique de la maladie. Nous avons développé une nouvelle approche de capture magnétique sur une puce microfluidique, basée sur l’assemblage de nanoparticules magnétiques (MNP) en suspension pour une séparation magnétique efficace dans les biofluides. Cette technologie innovante a servi d'élément crucial au sein d'un processeur fluidique, automatisant la séparation des VE du sang total basée sur la taille et l'affinité. Au-delà de la simplification du processus, elle améliore considérablement la reproductibilité et la répétabilité de la purification des exosomes. Cette thèse présente une approche multidisciplinaire pour améliorer le pronostic du cancer. Les résultats offrent un premier aperçu du profil transcriptomique des exosomes dans le sang des patients atteints de B-ALL, révélant des biomarqueurs potentiels spécifiques au sous-type. Le dispositif microfluidique répond à un besoin pressant en permettant une purification efficace des VE à partir du sang total, prometteur pour l'application clinique. / B-cell acute lymphoblastic leukemia (B-ALL) stands as the foremost cause of mortality due to disease in children. While modern chemotherapy can cure a majority of patients, around 20% exhibit inadequate response or are susceptible to relapse. Stratifying patients into distinct molecular subtypes through cytogenetics, molecular biology, and transcriptomic profiling is essential for tailoring risk-oriented therapies. However, the time required for traditional tumor sample analysis hampers swift intervention at the onset of treatment. As an alternative to conventional tissue biopsies, we proposed that extracellular vesicles (EVs), notably exosomes present in peripheral blood, are potential carriers of RNA-based biomarkers of B-ALL. Through analysis of a patient cohort representing two distinct molecular B-ALL subtypes, exosome-encapsulated RNA transcripts emerged as promising non-invasive biomarkers. These findings advocate for the early detection and ongoing monitoring of childhood B-ALL using exosome-based liquid biopsies. Nonetheless, the lengthy and intricate exosome purification process necessitates standardization and enhancement to establish credibility in clinical disease monitoring. We then developed a novel magnetic capture approach on a microfluidic chip, employing suspended magnetic nanoparticle (MNP) assemblies for efficient in-flow magnetic separation in biofluids. This innovative technology served as a crucial element within a fluidic processor, automating the purification of exosomes from whole blood through size- and affinity-based separation. Beyond simplifying the process, it significantly heightens the reproducibility and consistency of exosomes purification. This thesis presents a multifaceted approach to enhance cancer prognosis. The results offer a first insight into the transcriptomic landscape of exosomes in the blood of B-ALL patients, revealing potential subtype-specific biomarkers. The microfluidic device addresses a pressing need by enabling efficient exosome purification from whole blood, holding promise for clinical translation.

Cancer progression is accompanied with alterations in the cell biomechanical phenotype, including changes in cell structure, morphology, and responses to microenvironmental stress. These alterations result in an increased deformability of transformed cells and reduced resistance to mechanical stimuli, enabling motility and invasion. Therefore, single cell biomechanical properties could be served as a powerful label-free biomarker for effective characterization and early detection of single cancer cells. Advances and innovations in microsystems and nanotechnology have facilitated interrogation of the biomechanical properties of single cells to predict their tumorigenicity, metastatic potential, and health state. This dissertation utilized Atomic Force Microscopy (AFM) for the cell biomechanical phenotyping for cancer diagnosis and early detection, efficacy screening of potential chemotherapeutic agents, and also cancer stem-like/tumor initiating cells (CSC/TICs) characterization as the critical topics received intensive attention in the search for effective cancer treatment. Our findings demonstrated the capability of exogenous sphingosine to revert the aberrant biomechanics of aggressive cells and showed a unique, mechanically homogeneous, and extremely soft characteristic of CSC/TICs, suitable for their targeted isolation. To make full use of cell biomechanical cues, this dissertation also considered the application of nonlinear viscoelastic models such as Fractional Zener and Generalized Maxwell models for the naturally complex, heterogeneous, and nonlinear structure of living cells. The emerging need for a high-throughput clinically relevant alternative for evaluating biomechanics of individual cells led us to the development of a microfluidic system. Therefore, a high-throughput, label-free, automated microfluidic chip was developed to investigate the biophysical (biomechanical-bioelectrical) markers of normal and malignant cells. Most importantly, this dissertation also explored the biomechanical response of cells upon a dynamic loading instead of a typical transient stress. Notably, metastatic and non-metastatic cells subjected to a pulsed stress regimen exerted by AFM exhibited distinct biomechanical responses. While non-metastatic cells showed an increase in their resistance against deformation and resulted in strain-stiffening behavior, metastatic cells responded by losing their resistance and yielded slight strain-softening. Ultimately, a second generation microfluidic chip called an iterative mechanical characteristics (iMECH) analyzer consisting of a series of constriction channels for simulating the dynamic stress paradigm was developed which could reproduce the same stiffening/softening trends of non-metastatic and metastatic cells, respectively. Therefore, for the first time, the use of dynamic loading paradigm to evaluate cell biomechanical responses was used as a new signature to predict malignancy or normalcy at a single-cell level with a high (~95%) confidence level. / Ph. D.

Bioimpedance presents a versatile, label-free means of monitoring biological cells and their responses to physical, chemical and biological stimuli. Breast cancer is the second most common type of cancer among women in the United States. Although significant progress has been made in diagnosis and treatment of this disease, there is a need for robust, easy-to-use technologies that can be used for the identification and discrimination of critical subtypes of breast cancer in biopsies obtained from patients. This dissertation makes contributions in three major areas towards addressing the goal. First, we developed miniaturized bioimpedance sensors using MEMS and microfluidics technology that have the requisite traits for clinical use including reliability, ease-of-use, low-cost and disposability. Here, we designed and fabricated two types of bioimpedance sensors. One was based on electric cell-substrate impedance sensing (ECIS) to monitor cell adhesion based events and the other was a microfluidic device with integrated microelectrodes to examine the biophysical properties of single cells. Second, we examined a panel of triple negative breast cancer (TNBC) cell lines and a hormone therapy resistant model of breast cancer in order to improve our understanding of the bioimpedance spectra of breast cancer subtypes. Third, we explored strategies to improve the sensitivity of the microelectrodes to bioimpedance measurements from breast cancer cells. We investigated nano-scale coatings on the surface of the electrode and geometrical variations in a branched electrode design to accomplish this. This work demonstrates the promise of bioimpedance technologies in monitoring diseased cells and their responses to pharmaceutical agents, and motivates further research in customization of this technique for use in personalized medicine. / Ph. D.

Die Fähigkeit künstlicher Mikroschwimmer, auf äußere Reize zu reagieren und deren mechanistische Ursprünge, gehören zu den umstrittensten Fragen der interdisziplinären Wissenschaft. Die Erzeugung chemischer Gradienten ist dabei eine technische Herausforderung, da sie aufgrund von Diffusion schnell abflachen. Inspiriert von ‘Stop-flow’ Experimenten aus der chemischen Kinetik zeigen wir, dass die Erzeugung eines mikrofluidischen Gradienten durch Kombination mit einer Druckrückkopplungsschleife zur präzisen Kontrolle des Stoppens erfolgen kann. Das ermöglicht es uns, die mechanistischen Details der Chemotaxis von künstlichen katalytischen Janus-Mikromotoren zu untersuchen. Wir stellen fest, dass diese Kupfer-Janus-Partikel eine chemotaktische Bewegung entlang des Konzentrationsgradienten sowohl in positiver als auch in negativer Richtung zeigen, und wir demonstrieren die mechanische Reaktion der Partikel auf unausgewogene Widerstandskräfte, die dieses Verhalten erklären.