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Supported Lipid Bilayer As a Biomimetic Platform for Neuronal Cell Culture

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Supported Lipid Bilayer As a Biomimetic Platform for Neuronal Cell Culture Supported lipid bilayer as a biomimetic platform for neuronal cell culture Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Master of Science Dzmitry Afanasenkau aus Zhodzina, Weißrussland Berichter: Universitätsprofessor Dr. rer. nat. habil. Andreas Offenhäusser Universitätsprofessor Dipl. Ing. Dr. Werner Baumgartner Tag der mündlichen Prüfung: 20.12.2012 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar ii Forschungszentrum Jülich GmbH Peter Grünberg Institute / Institute of Complex Systems (PGI / ICS) Bioelectronics (PGI-8 / ICS-8) Supported lipid bilayer as a biomimetic platform for neuronal cell culture Dzmitry Afanasenkau Schriften des Forschungszentrums Jülich Reihe Information / Information Band / Volume 26 ISSN 1866-1777 ISBN 978-3-89336-863-1 Bibliographic information published by the Deutsche Nationalbibliothek. The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at http://dnb.d-nb.de. Publisher and Forschungszentrum Jülich GmbH Distributor: Zentralbibliothek 52425 Jülich Tel: +49 2461 61-5368 Fax: +49 2461 61-6103 Email: [email protected] www.fz-juelich.de/zb Cover Design: Grafische Medien, Forschungszentrum Jülich GmbH Printer: Grafische Medien, Forschungszentrum Jülich GmbH Copyright: Forschungszentrum Jülich 2013 Schriften des Forschungszentrums Jülich Reihe Information / Information, Band / Volume 26 D 82 (Diss., RWTH Aachen, Univ., 2012) ISSN 1866-1777 ISBN 978-3-89336-863-1 The complete volume is freely available on the Internet on the Jülicher Open Access Server (JUWEL) at www.fz-juelich.de/zb/juwel Neither this book nor any part of it may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Contents List of abbreviations ..............................................................................................vii List of figures...........................................................................................................ix List of tables..........................................................................................................xiii Abstract....................................................................................................................1 Zusammenfassung...................................................................................................3 Chapter 1: Fundamentals.......................................................................................5 1.1 The cell membrane..........................................................................................5 1.1.1 Cells and membranes...............................................................................5 1.1.2 The structure of the cell membrane.........................................................5 1.1.3 Functions of the cell membrane.............................................................10 1.1.4 Membrane potential...............................................................................11 1.1.5 Surface charge of the cell membrane.....................................................13 1.1.6 Electrical double layer...........................................................................13 1.2 Physics of the lipid bilayer............................................................................15 1.2.1 Hydrophobic interactions.......................................................................15 1.2.2 Lipid polymorphism...............................................................................15 1.2.3 Lipid movements in the bilayer.............................................................17 1.3 Artificial lipid membrane systems................................................................19 1.3.1 Lipid vesicles........................................................................................19 1.3.2 Langmuir monolayers............................................................................20 1.3.3 Suspended lipid bilayers........................................................................21 1.3.4 Supported lipid bilayers (SLB)..............................................................22 1.4 Cell adhesion and signalling.........................................................................24 1.4.1 Membrane receptors...............................................................................24 1.4.2 Signalling via adhesion molecules.........................................................25 1.4.3 Eph-ephrin signaling..............................................................................26 Chapter 2: Motivation and goals.........................................................................29 2.1Interfaces in nature and technology...............................................................29 2.2Why SLB?......................................................................................................30 iii 2.2.1Advantages of the SLB...........................................................................30 2.2.2Applications of the SLB..........................................................................31 2.2.3SLB for cell culture ................................................................................31 2.3The aim and directions of work.....................................................................32 2.3.1The aim...................................................................................................32 2.3.2Increasing cell adhesion to the SLB........................................................33 2.3.3SLB with incorporated ephrin A5...........................................................33 2.3.4Preparation of patterned SLB using GUV..............................................34 Chapter 3: Materials and methods......................................................................35 3.1 Preparation of the supported lipid bilayers...................................................35 3.1.1 Overview of the preparation procedure.................................................35 3.1.2 Substrate preparation.............................................................................35 3.1.3 Preparation of small unilamellar vesicles (SUV)...................................36 3.1.4 Formation of supported lipid bilayers ...................................................37 3.1.5 Lipids ....................................................................................................37 3.2 Incorporation of proteins in to SLB..............................................................39 3.2.1 Overview of the protein incorporation procedure..................................39 3.2.2 Determination of the concentration of detergent...................................41 3.3 Optical methods............................................................................................42 3.3.1 Microscopy............................................................................................42 3.3.2 Fluorescence recovery after photobleaching (FRAP)............................42 3.4 Cell culture....................................................................................................45 3.4.1 Preparation and plating of neurons........................................................45 3.4.2 Preparation of poly-L-lysine coated glass..............................................46 3.4.3 Cell counting and staining.....................................................................46 3.5 QCM-D.........................................................................................................48 3.6 Nanoparticle Tracking Analysis....................................................................51 3.7 Patch-clamp...................................................................................................52 3.8 Immunofluorescence of lipid bilayers..........................................................55 3.9 Preparation of GUV......................................................................................56 3.10 Protein microcontact printing.....................................................................57 Chapter 4: Characterization of liposomes and SLBs........................................59 4.1Characterization of liposomes........................................................................59 4.2Characterization of SLB................................................................................60 4.2.1QCM-D measurements...........................................................................60 4.2.2Observations using fluorescent microscopy...........................................62 4.2.3FRAP.......................................................................................................62 Chapter 5: Positively charged SLB for neuronal ..............................................67 5.1Adhesion of neurons to charged SLB ...........................................................67 5.1.1Observations of neurons on charged SLB..............................................67 iv 5.1.2Dependence of cell adhesion and growth on charge density..................69 5.1.3Discussion of adhesion and growth of neurons on SLBs.......................73 5.2Electrophysiology of cells
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