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Dissertation Submitted to the Combined Faculty of Natural Dissertation submitted to the Combined Faculty of Natural Sciences and Mathematics of the Ruperto Carola University Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by M.Sc. Elena von Molitor born in: Sinsheim Oral examination: 10.05.2021 Human tongue-cell derived spheroids suggest that multiple pathways and molecules are involved in the sweet gustatory sense Referees: Prof. Dr. Stephan Frings Referees: Prof. Dr. Rüdiger Rudolf I dedicate this thesis to my parents and my family who always encouraged me to go on adventures. Thank you for your endless love and support. Table of contents Table of contents Acknowledgements ............................................................................................................. V Zusammenfassung ............................................................................................................ VII Abstract............................................................................................................................... IX 1 Introduction .................................................................................................................. 9 1.1 Taste papillae and their projections to the central nervous system .......................... 2 1.2 Taste buds and their composition ............................................................................ 3 1.3 The canonical sweet taste receptor is formed by T1R2/T1R3 GPCR heterodimers ... 4 1.4 Multiple G-protein subunits may transduce sweet taste ........................................... 5 1.5 Canonical sweet taste transduction downstream the sweet taste receptor .............. 6 1.6 Caloric sugars may signal in a manner independent of the sweet taste receptor ..... 8 1.7 The sweet taste receptor-independent pathway may mediate cephalic phase Insulin release ........................................................................................................12 1.8 GLP-1 released from taste cells may exert an endocrine action on target tissues ....13 1.9 The sweet taste receptor-independent pathway may be differently decoded in the central nervous system than the canonical pathway .........................................14 1.10 The sweet taste receptor-independent pathway may prepare the body for digestion ................................................................................................................16 1.11 Sweet taste signaling is not restricted to the oral cavity ..........................................17 1.12 The lack of adequate test systems calls for the development of new taste models ...19 1.13 Molecular sensors to study sweet taste signaling ...................................................22 1.14 Human fungiform papillae-derived taste cells resemble type II bitter-sensitive cells .......................................................................................................................23 1.15 The potential of 3D cell cultures .............................................................................25 1.16 3D cell culture in practice .......................................................................................28 1.17 Aim of this study .....................................................................................................29 2 Methods .......................................................................................................................31 2.1 Cell culture media ..................................................................................................31 2.2 Cell culture and expansion .....................................................................................32 2.3 Generation of 3D taste cultures ..............................................................................32 I Table of contents 2.4 Immunostaining protocols.......................................................................................33 2.5 Optical clearing protocols .......................................................................................36 2.6 Live cell imaging protocols .....................................................................................38 2.7 Transcriptome analysis of spheroids ......................................................................41 2.8 Statistics and software ...........................................................................................42 3 Results .........................................................................................................................43 3.1 Establishment of an optical clearing protocol for HTC-8 3D cultures ......................43 3.2 Characterization of HTC-8 3D cultures ...................................................................46 3.3 The lack of reliable antibodies urges the need of a functional Ca2+ assay to study gustatory responses of HTC-8 spheroids ......................................................49 3.4 Development of a perfused live cell imaging setup for confocal microscopy ...........50 3.5 Analysis of Saccharin-induced Ca2+ transients in HTC-8-G-GECO spheroids .......53 3.6 Analysis of ATP-induced Ca2+ transients in HTC-8-G-GECO spheroids .................55 3.7 Compound diffusion into HTC-8-G-GECO spheroids induced a delay in Ca2+ transients .......................................................................................................57 3.8 Development of a perfused live cell imaging setup for LSFM .................................59 3.9 HTC-8-G-GECO cells responded to sugars only in 3D culture ...............................62 3.10 Analysis of Sucrose-induced Ca2+ transients in HTC-8-G-GECO spheroids ...........64 3.11 Analysis of KCl-induced Ca2+ transients in HTC-8-G-GECO spheroids ..................66 3.12 Progenitor cells as a promising source to gain sweet-sensitive cells ......................68 4 Discussion ...................................................................................................................71 4.1 Optical clearing with Glycerol allows penetration deep into HTC-8 spheroids .........72 4.2 Spheroids and Dynarray chips are feasible models to generate in vitro taste bud-like structures ..................................................................................................73 4.3 Perfused live cell imaging systems mimic the physiological application of compounds and allow the analysis of acute gustatory responses ...........................74 4.4 Sweet and bitter responses follow different kinetics in HTC-8-G-GECO spheroids ...76 4.5 Saccharin has sweet and bitter taste ......................................................................78 4.6 Bitter compounds may activate the canonical pathway in HTC-8-G-GECO spheroids ...............................................................................................................79 II Table of contents 4.7 Sugars may activate the sweet taste receptor-independent pathway in HTC-8- G-GECO spheroids ................................................................................................86 4.8 Possible ways for Ca2+ entry in HTC-8-G-GECO spheroids ....................................90 4.9 Purinergic intercellular signaling may boost and/or transmit taste responses in HTC-8-G-GECO spheroids ....................................................................................91 4.10 HTC-8-G-GECO spheroids may contain tightly and broadly tuned cells .................94 4.11 Further perspectives ...............................................................................................95 4.12 Outlook: HTP-76 cells as a potential unlimited cell source to study taste cell differentiation .........................................................................................................97 4.13 Conclusion .............................................................................................................99 5 References ................................................................................................................. 101 6 Appendix .................................................................................................................... 133 6.1 List of abbreviations ............................................................................................. 133 6.2 List of figures ........................................................................................................ 136 6.3 List of tables ......................................................................................................... 137 6.4 Supplementary data ............................................................................................. 138 7 Publications ............................................................................................................... 143 8 Statement on copyright and self-plagiarism ........................................................... 144 III Acknowledgements Acknowledgements First and foremost, I owe my deepest gratitude Prof. Dr. Rüdiger Rudolf. Thank you for all your expert advises, your dedicated support and patience. I appreciated that you always had time to listen to my struggles and I am especially grateful for your courage and trust in my work. I absolutely enjoyed working in your lab and your enthusiasm about science always motivated me to stay tuned. This allowed me not only to gain scientific experience but also to grow personally. Many thanks go to Prof. Dr. Mathias Hafner for his guidance and overall insights in the cell biology field which have made this an inspiring experience
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