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Microfabrication and Microfluidics for 3D Brain-On-Chip

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Microfabrication and Microfluidics for 3D Brain-On-Chip Microfabrication and microfluidics for 3D brain-on-chip Bart Schurink Microfabrication and microfluidics for 3D brain-on-chip Bart Schurink i Members of the committee: Chairman Prof. dr. ir. J.M.W. Hilgenkamp (voorzitter) Universiteit Twente Promotor Prof. dr. J.G.E. Gardeniers Universiteit Twente Co-promotor/supervisor Dr. R. Luttge Universiteit Eindhoven Members Prof. dr. J.C.T. Eijkel University of Twente Prof. dr. H.B.J. Karperien University of Twente Prof. dr. A.S. Holmes Imperial College London Prof. dr. ir. J.M.J. den Toonder Technical University of Eindhoven Prof. dr. ir. R. Dekker Delft University of Technology The work in this thesis was carried out in the Mesoscale Chemical Systems group and MESA+ Institute for Nanotechnology, University of Twente. It is part of the MESOTAS project financially supported by ERC, Starting Grant no. 280281 Title: Microfabrication and microfluidics for 3D brain-on-chip Author: Bart Schurink ISBN: 978-90-365-4142-8 DOI: 10.3990/1.9789036541428 Publisher: Gildeprint, Enschede, The Netherlands Copyright © 2016 by Bart Schurink, Enschede, The Netherlands. No part of the this book may be copied, photocopied, reproduced, translated or reduced to any electronic medium or machine-readable form, in whole or in part, without specific permission of the author. ii Microfabrication and microfluidics for 3D brain-on-chip PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit Twente, op gezag van de rector magnificus, Prof. dr. H. Brinksma, volgens besluit van het College voor Promoties in het openbaar te verdedigen op donderdag 23 juni 2016 door Bart Schurink Geboren op 6 november 1982 te Oldenzaal, Nederland iii Dit proefschrift is goedgekeurd door de promotor: Prof. dr. J.G.E. Gardeniers Co-promotor/supervisor: Dr. R. Luttge iv Contents 1. Introduction 1 1.1 Brain-on-chip 2 1.2 Background: investigating brain functions 2 1.3 Technical measurement concepts in neurology 3 1.3.1 Optical imaging 4 1.3.2 Patch clamping 4 1.3.3 Intracellular measurements 5 1.3.4 Extracellular measurements 5 1.4 Extracellular recordings by microelectrode arrays (MEA) 6 1.5 Improving the conventional MEA 7 1.6 Novel concepts: organ-on-chip 8 1.7 Aim of this work: microfabrication technology for brain-on-chip 9 1.8 Thesis outline 10 1.9 References 11 2. Advances in 3D neuronal cell culture 15 2.1 Introduction 16 2.2 Materials and methods 19 2.2.1 Single cell seeding in 3D pores 19 2.2.2 Cell fixation and preparation for SEM 20 2.2.3 Nanoscaffold fabrication 20 2.2.4 Cell culture and staining 20 2.2.5 Matrix preparation and analysis 21 2.2.6 Decellularized ECM for neuronal cell culture 22 2.3 Results and discussion 23 2.3.1 Single cell seeding in 3D pores 23 2.3.2 The impact of surface nanogrooves on 3D cell alignment 24 2.3.3 Neuronal cells inside 3D matrices 25 2.3.4 Decellularized ECM for neuronal cell culture 27 2.4 Conclusions 29 2.5 References 29 3. A hydrogel/PDMS hybrid bioreactor facilitating 3D cell culturing 33 3.1 Introduction 34 v 3.2 Methods and materials 36 3.2.1 Bioreactor fabrication 36 3.2.2 Transport through the barrier 37 3.2.3 Cell culture in the bioreactor 38 3.3 Results 39 3.3.1 Bioreactor fabrication 39 3.3.2 Transport through the barrier 39 3.3.3 Cell culture in the bioreactor 40 3.4 Discussion 42 3.5 Conclusions 43 3.6 References 43 4. Bioreactor coupled capillary electrophoresis for on-chip measurement of 45 cell culture metabolites 4.1 Introduction 46 4.2 Materials and Methods 47 4.2.1 Measurement of Lactate using CE-minilab 47 4.2.2 Integration of microbioreactor 48 4.2.3 On-chip measurements 49 4.3 Results and discussion 50 4.3.1 Measurement of Lactate using CE-minilab 50 4.3.2 Integration of microbioreactor 52 4.3.3 On-chip measurements 53 4.4 Conclusions 53 4.5 References 54 5. Highly uniform sieving structure by corner lithography and silicon wet 57 etching 5.1 Introduction 58 5.2 Design of silicon sieves with highly uniform 3D pores 59 5.2.1 Sieve area, thickness and pore geometry 59 5.2.2 Design criteria to compensate for the non-uniformity of the sieve 60 thickness 5.3 Materials and methods 62 5.3.1 Sieve fabrication process applying corner lithography and back-etch 62 vi 5.3.2 Selection of an etchant for the deep anisotropic back-etch process 63 5.3.3 Analysis methods 64 5.4 Results and discussion 64 5.4.1 Realization of pyramidal pits and octahedral structures 64 5.4.2 Back-etch performance using KOH 65 5.4.3 Optimizing deep anisotropic silicon back-etching 68 5.5 Conclusions 71 5.6 Supporting information 72 5.7 References 74 6. Microsieve-electrode array fabrication 77 6.1 Introduction 78 6.2 Materials and Methods 80 6.2.1 Patterning of the electrode material 80 6.2.2 Doping of the poly-silicon 80 6.2.3 Patterning of the SiRN isolation layer 81 6.2.4 Formation of low resistivity TiSi2 on the sensing electrodes and 84 dicing 6.3 Results and Discussion 85 6.3.1 Patterning of the electrode material 85 6.3.2 Doping of the poly-silicon 86 6.3.3 Patterning of the SiRN isolation layer 86 6.3.4 Formation of low resistivity TiSi2 on the sensing electrodes and 89 dicing 6.4 Conclusions 93 6.5 Supporting information 93 6.6 References 98 7. Microsieve electrode array characterization 101 7.1 Introduction 102 7.2 Material and methods 105 7.2.1 Titanium nitride as alternative electrode material 105 7.2.2 Size and shape of the poly-Si sensing electrode in the μSEA 106 7.2.3 μSEA electrical characterization 108 7.2.4 Positioning of μSEA transducer neurons 109 vii 7.2.5 Culturing and analysis of μSEA transducer neurons 110 7.3 Results and discussion 111 7.3.1 Titanium nitride as alternative electrode material 111 7.3.2 Size and shape of the poly-Si sensing electrode in the μSEA 113 7.3.3 μSEA electrical characterization 114 7.3.4 Positioning of μSEA transducer neurons 117 7.3.5 Culturing and analysis of μSEA transducer neurons 118 7.4 Conclusions 120 7.5 References 121 8. Future applications of brain on Chip technology 125 8.1 Introduction 126 8.2 Impact of Brain on Chip technology 127 8.2.1 The neuro(electro)physiology in 3D cultured neuronal networks is 127 different 8.2.2 Organ-on-chips as clinical human models of the future 128 8.2.3 Brain-on-chip 128 8.2.4 Blood-Brain-Barrier models (BBB) 129 8.2.5 Acute tissue slices 130 8.3 μSEA applications and state of the art neuro-assays 132 8.3.1. Neurodynamics and models for axon injury and neurotoxicity 132 8.3.2 Stacking MEAs 135 8.4 Conclusions 136 8.5 References 137 9. Conclusions and Outlook 139 9.1 Conclusions 140 9.2 Outlook 141 9.3 References 143 Appendix: Process flow 145 List of publications 173 Summary 175 Samenvatting 177 Dankwoord 181 viii 1 Introduction This chapter describes the background and goals of a brain-on-chip and outlines the challenges of the project: The terminology, definitions and state-of-the-art of deciphering brain functions outside of a living organism are discussed and an overview of current technical concepts, tools and applications are given. Finally, an outline of this thesis is presented providing a short description of the contents of each chapter. Chapter 1 1.1 Brain-on-chip The work described in this thesis has been performed within the larger framework of the ”MESOTAS” project (grant number 280281) which is financed by the European Research Council (ERC). MESOTAS stands for Mesoscale Total Analysis Systems, which can be utilized for the investigation of miniaturized, three-dimensional cell cultures. Since the scope of this PhD project is microsystems technology for brain-on- chip, here, the focus is on the improvement of existing culture approaches for the study of neuronal tissue in-vitro, combining recent developments in micro and nanotechnology, tissue engineering, microfluidics and neuroelectrophysiology. The research was carried out at Mesoscale Chemical Systems (MCS) in affiliation with the MESA+ Institute for Nanotechnology of the University of Twente, The Netherlands and the Microsystems group at the Mechanical Engineering department of Eindhoven University of Technology, The Netherlands. First, the following sections sketch the multidisciplinary character of this PhD project and introduce the reader to the common terminology used in this field of research. Subsequently, in the last two sections the aim and the outline of this thesis are summarized. 1.2 Background: investigating brain functions The most complex organ of our body, is the brain, which is part of the central nervous system. Basically, the brain acts as a central processing unit (CPU) for obtaining, analyzing and storing data in the biological domain. The brain consists of the cerebrum, composed of the right and left hemispheres enabling higher cognitive functions, the cerebellum, coordinating muscle movement, and the control center called the brainstem. For this project, we are mainly interested in the surface of the cerebrum, the cortex, which contains the majority of nerve cells.1 In ancient times, the brain was solely seen as “cranial stuffing” and the heart was the center of intelligence. Many years later, during the Roman times, this believe was corrected.
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