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

We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 µm. The diffusive limit of competition for media occurred with a pitch of ≥1250 µm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

In novel drug delivery system, the encapsulation of therapeutic cells in microparticles has great promises for the treatment of a range of health con- ditions. Therefore, the encapsulation material and technology are of great importance to the validity and efficiency of the advanced medical therapy. Several unsolved challenges in regards to versatile microparticle synthesis ma- terials and methods form the main obstacle for a translation of novel cell therapy concepts from research to clinical practice. Thiol-ene based polymer systems have emerged and gained great popular- ity in material development in general and in biomedical applications specif- ically. The thiol-ene platform is broad and therefore of interest for a variety of applications. At the same time, many aspects of this material platform are largely unexplored, for example material and manufacturing technology developments for microfluidic applications . In this Ph.D. thesis, thiol-ene materials are explored for use in cell encap- sulation. The marriage of these two technology fields breeds the possibility for a novel microfluidic cell encapsulation approach using a novel encapsulation material. To this end, several new manufacturing technologies for thiol-ene and thiol-ene-epoxy droplet microfluidic devices were developed. Moreover, core-shell microparticle synthesis for cell encapsulation based on a novel co- synthesis concept using a thiol-ene based material was developed and inves- tigated. Finally, a thiol-ene-epoxy system was also used for the formation of microwells and microchannels that improve protein analysis on microarrays. The first part of the thesis presents the background and state-of-the-art technologies in regards to cell therapy, microfluidics, and thiol-ene based ma- terials. In the second part of the thesis, a novel manufacturing approach of thiol-ene-epoxy material as well as core-shell particle co-synthesis in micro- fluidics using thiol-ene based material are presented and characterized. The third part of the thesis presents the cell viability studies of encapsulated cells using the novel encapsulation material and method. In the final part of the thesis, two applications of thiol-ene-epoxy gaskets for protein detection mi- croarrays are presented. / Inkapsling av levande celler i mikrokapslar för terapeutiska ändamål är mycket lovande för frmatida behandling av många olika sjukdomar. Emeller- tid är en behandlings effektivitet i hög grad beroende av vilka material som används för inkapsling och vilken teknisk lösning som används för att ska- pa mikrokapslarna. För närvarande återstår det många utmaningar för att omvandla grundforskningresultat till klinisk verklighet, vilken kräver mer än- damålsenliga tillvägagångssätt för att tillverka mikrokapslar i material som är kompatibla med användningsområdena. De senaste åren har tiol-en baserade polymerer har blivit mycket använda för materialutveckling i stort och för biomedicinska tillämpningar i synnerhet. Med tiol-en kemi kan en mycket stor mängd helt olika syntetiska material framställas, vilket gör tiol-ener intressanta för en mängd applikationer. För närvarande är dock mycket inom denna materialklass outforskat, t.ex. inom material och tillverkningmetodik för mikrofluidiktillämpningar. I denna avhandling används tiol-ener för cellinkapsling. Sammanslagning av dessa teknologier möjliggör en ny typ av cellinkapsling med nya materi- alegenskaper. En mängd olika tillverkningssätt där tiol-en eller tiol-en-epoxi används för droplet-mikrofluidiksystem utvecklades. Core-shell mikrokapsel- syntes för cell-inkapsling baserat på en ny metod för samtidig syntes av både core och shell utvecklades och karaktäriserades. Slutligen utvecklades ett tiol- en-epoxi system för enkel integrering med proteinmikroarrayer på objektsglas. I avhandlingens första del presenteras bakgrund och dagens bästa teknolo- gier för terapeutisk cellinkapsling, mikrofluidik och tiol-en baserade material. I avhandlingens andra del presenteras en ny tillverkningsmetod för mikro- strukturerade tiol-en-epoxi artiklar och samtidig syntes av core och shell för mikrokapslar med användande av mikrofluidik. I den tredje delen presenteras cellöverlevandsstudier för de celler som inkapslats med de nya materialen och de nyutvecklade metoderna. I den avslutande delen beskrivs två specifika fall där tiol-en-epoxi komponenter används för proteindetektion och mikroarrayer. / <p>QC 20171122</p>

We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 µm. The diffusive limit of competition for media occurred with a pitch of ≥1250 µm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.