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Development of Advanced Human Intestinal in Vitro Models *** Entwicklung Von Erweiterten Humanen Intestinalen in Vitro Modellen

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Development of Advanced Human Intestinal in Vitro Models *** Entwicklung Von Erweiterten Humanen Intestinalen in Vitro Modellen Development of advanced human intestinal in vitro models *** Entwicklung von erweiterten humanen intestinalen in vitro Modellen Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences, Julius-Maximilians-Universität Würzburg, Section Clinical Sciences submitted by Matthias Oliver Schweinlin from Lörrach Würzburg 2016 Submitted on: …………………………………………………………..…….. Members of the Promotionskomitee: Chairperson: Prof. Dr. Thomas Hünig Primary Supervisor: Prof. Dr. Heike Walles Supervisor (Second): Prof. Dr. Stefan Störk Supervisor (Third): PD Dr. Beate Niesler Date of Public Defence: …………………………………………….………… Date of Receipt of Certificates: ………………………………………………. Table of contents Table of contents List of figures ............................................................................................................................................. IV List of tables ............................................................................................................................................... VI Abbreviations .......................................................................................................................................... VII Summary ..................................................................................................................................................... XI Zusammenfassung ................................................................................................................................ XIII 1 Introduction ....................................................................................................................................... 1 1.1 The small intestine ................................................................................................................... 1 1.1.1 Anatomy and physiology of the small intestine ................................................... 1 1.1.2 The intestinal mucosa - cell types and molecular regulation ......................... 2 1.1.3 Gut homeostasis - regulation/dysregulation ........................................................ 7 1.2 The gut barrier - intestinal transport mechanisms ..................................................... 8 1.2.1 Paracellular transport .................................................................................................... 9 1.2.2 Transcellular passive/active transport................................................................... 9 1.2.3 Efflux transport ............................................................................................................. 10 1.2.4 Endocytosis ..................................................................................................................... 10 1.3 Preclinical model systems .................................................................................................. 11 1.3.1 In vivo ................................................................................................................................ 11 1.3.2 Ex vivo ............................................................................................................................... 11 1.3.3 In vitro .............................................................................................................................. 12 1.3.4 In silico modelling ........................................................................................................ 16 1.4 Tissue engineering ................................................................................................................ 17 1.5 Applications of peroral test systems in biomedical research .............................. 18 2 Scope of thesis ................................................................................................................................ 20 3 Materials ........................................................................................................................................... 21 3.1 Biological materials .............................................................................................................. 21 3.2 Equipment ................................................................................................................................ 21 3.3 Disposable materials ............................................................................................................ 23 3.4 Laboratory materials ........................................................................................................... 24 3.5 Chemicals and solutions ..................................................................................................... 25 3.6 General buffers and solutions for cell culture ............................................................ 30 3.7 Cell culture medium ............................................................................................................. 30 Table of contents 3.8 Materials and solutions for histology ............................................................................ 32 3.9 Enzymes .................................................................................................................................... 33 3.10 Antibodies ............................................................................................................................ 33 3.11 qPCR ....................................................................................................................................... 34 3.12 Software ................................................................................................................................ 34 4 Methods............................................................................................................................................. 36 4.1 Cell culture ............................................................................................................................... 36 4.1.1 Preparation of porcine decellularized scaffolds ............................................... 36 4.1.2 Caco-2 cell line ............................................................................................................... 37 4.1.3 Dendritic cells ................................................................................................................ 38 4.1.4 Primary cells from human intestinal tissue - isolation and expansion ... 39 4.1.5 Wnt3A-conditioned medium ................................................................................... 40 4.1.6 Primary intestinal tissue model - different culture conditions .................. 40 4.2 Histology ................................................................................................................................... 41 4.2.1 Fixation and embedding of tissues ........................................................................ 41 4.2.2 Deparaffinization and rehydration of tissue sections .................................... 42 4.2.3 Haematoxylin & Eosin (H&E) staining ................................................................. 43 4.2.4 Feulgen staining ............................................................................................................ 44 4.2.5 Masson’s trichrome staining .................................................................................... 44 4.2.6 Immunofluorescence ................................................................................................... 45 4.2.7 SEM and TEM imaging ................................................................................................ 46 4.3 Flow cytometry ...................................................................................................................... 46 4.4 Gene expression analysis by qPCR ................................................................................. 47 4.5 FITC-dextran assay ............................................................................................................... 47 4.6 TEER-measurement ............................................................................................................. 48 4.7 Transport studies .................................................................................................................. 49 4.8 Nanoparticles .......................................................................................................................... 49 5 Results ............................................................................................................................................... 51 5.1 Functional tests on an improved Caco-2 model ........................................................ 51 5.1.1 Adaption of the Caco-2 model.................................................................................. 51 5.1.2 Immune cell differentiation ...................................................................................... 52 5.1.3 Caco-2 transport assay performance .................................................................... 54 5.2 Establishment of a primary small intestinal tissue model .................................... 61 Table of contents 5.2.1 Pilot studies for model establishment .................................................................. 61 5.2.2 Adaption and detailed characterization of the primary model .................. 68 5.2.3 Influence of controlled dynamic culture conditions ....................................... 70 5.2.4 Molecular characterization ......................................................................................
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