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© Copyright 2020 Tianzi Zhang © Copyright 2020 Tianzi Zhang Studying Intercellular Signaling Underlying Human Diseases Using Open Microfluidic Coculture Tianzi Zhang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2020 Reading Committee: Ashleigh B. Theberge, Chair Robert E. Synovec Jesse G. Zalatan Program Authorized to Offer Degree: Chemistry University of Washington Abstract Studying Intercellular Signaling Underlying Human Diseases Using Open Microfluidic Coculture Tianzi Zhang Chair of the Supervisory Committee: Professor Ashleigh B. Theberge Department of Chemistry This dissertation focuses on the development of innovative open microfluidic cell culture platforms and their application in studying intercellular signaling underlying human diseases in controlled ex vivo microenvironments. Chapter 1 introduces the background of open microfluidic capillary systems, including the design considerations and current fabrication techniques, and addresses the advantages of using open microfluidic cell culture systems to study intercellular signaling. Chapter 2 presents a new open microfluidic capillary platform, which patterns biocompatible hydrogel walls along a rail insert set inside established cultureware. The permeable hydrogel walls provide segregation for the cells and support diffusion of soluble factors. Chapter 3 discusses a microscale collagen gel contraction assay with an engineered well plate insert that uses surface tension forces to load and manipulate small volumes of cell-laden collagen. The system is easily operated with two pipetting steps and the microscale device moves dynamically as a result of cellular forces. Chapter 4 presents an open microfluidic coculture platform consisting of two independent cell culture regions separated by a half wall. The cell types are selectively seeded into the regions and connected with cell culture media. Using the device, we show that human kidney tubular epithelial cells can tune organ-specificity in other endothelial cell types. Chapter 5 shows a preliminary asthma disease coculture model of human lung fibroblasts and primary alveolar macrophages using a similar open microfluidic coculture device to that described in Chapter 4. The preliminary cell culture result indicates that lung fibroblasts exhibit elevated cell contractility when cultured separately with alveolar macrophages in shared cell culture media. Chapter 6 concludes the thesis and proposes some future research directions and interesting metabolomic studies using existing or alternative open microfluidic systems. TABLE OF CONTENTS List of Figures ................................................................................................................................ vi Chapter 1. Introduction ....................................................................................................................1 1.1 Open Microfluidic Capillary Systems .............................................................................1 1.2 Design Considerations .....................................................................................................3 1.2.1 Conditions for SCF in an Open Channel ................................................................ 3 1.3 Open Microfluidic System Fabrication ............................................................................5 1.3.1 Fabrication Techniques ........................................................................................... 5 1.3.2 Surface Treatment ................................................................................................... 6 1.4 Key Advantages of Open Microfluidic Systems .............................................................7 1.4.1 Pipette Accessibility................................................................................................ 7 1.4.2 Imaging Capability.................................................................................................. 8 1.4.3 Design Flexibility.................................................................................................... 9 1.4.4 Open Microfluidics vs. Transwell Systems ............................................................ 9 1.4.5 Limitations and Areas for Improvement ............................................................... 10 1.5 Novel Advances of Open Microfluidic Tools in Biological Research ..........................11 1.5.1 Suspended Gel Patterning ..................................................................................... 11 1.5.2 Mechanobiology ................................................................................................... 12 1.5.3 Multi-Culture Platforms ........................................................................................ 13 1.6 References ......................................................................................................................14 Chapter 2. Upgrading Well Plates Using Open Microfluidic Patterning.......................................18 2.1 Abstract ..........................................................................................................................18 i 2.2 Introduction ....................................................................................................................19 2.3 Materials and Methods ...................................................................................................23 2.3.1 Devices .................................................................................................................. 23 2.3.2 Characterization of SCF ........................................................................................ 24 2.3.3 Confocal Imaging of Patterned Hydrogel Wall .................................................... 25 2.3.4 Cell Culture ........................................................................................................... 25 2.3.5 Determination of the Permeability of the Hydrogel Wall ..................................... 26 2.3.6 Validation of the Hydrogel Wall Integrity for Compartmentalized Cell Culture . 27 2.3.7 Comparison of LNCaP Morphology on Different Well Plate Surfaces ............... 28 2.3.8 Validation of the Modular Rail-Based Hydrogel Patterning Platform ................. 28 2.4 Results and Discussion ..................................................................................................30 2.4.1 Spontaneous Capillary Flow along a Rail System ................................................ 30 2.4.2 Design of the Open Microfluidic Hydrogel Patterning Platform.......................... 32 2.4.3 Application of Platform for Cell Culture Experiments ......................................... 34 2.5 Conclusion .....................................................................................................................38 2.6 Figures............................................................................................................................40 2.7 Supplementary Information ...........................................................................................46 2.8 References ......................................................................................................................54 Chapter 3. Investigating Fibroblast-Induced Collagen Gel Contraction Using a Dynamic Microscale Platform .......................................................................................................................59 3.1 Abstract ..........................................................................................................................59 3.2 Introduction ....................................................................................................................60 3.3 Materials and Methods ...................................................................................................62 ii 3.3.1 Devices Fabrication .............................................................................................. 62 3.3.2 Cell Culture ........................................................................................................... 63 3.3.3 HFL-1 Collagen Gel Contraction Assay in the CGC Device and 96 Well Plate .. 63 3.3.4 Measurement of CGC Device Angle .................................................................... 64 3.3.5 Viability Test ........................................................................................................ 65 3.3.6 Statistical Analysis ................................................................................................ 65 3.4 Results ............................................................................................................................66 3.4.1 Device Design and Workflow ............................................................................... 66 3.4.2 Viability Test ........................................................................................................ 68 3.4.3 FBS Augments Fibroblast Gel Contraction in Our Device .................................. 68 3.4.4 Eosinophils Cocultured With Fibroblasts Augment Collagen Gel Contraction in Our Device ............................................................................................................................ 69 3.5 Discussion ......................................................................................................................70 3.6 Figures............................................................................................................................74
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