Patch clamp experiments with human neuron-like cells under different gravity conditions Thesis presented in fulfilment of the thesis requirement for the degree of Doctor of Philosophy in Natural Sciences (Dr. rer. nat.) Faculty of Natural Sciences Universität Hohenheim Institute of Physiology (230) Department of Membrane Physiology (230b) presented by Florian Peter Michael Kohn from Stuttgart, Germany 2010 Dean: Prof. Dr. Heinz Breer Reviewer: Prof. Dr. Wolfgang Hanke Co-reviewer: Prof. Dr. Reinhardt Hilbig Submitted: 11th June 2010 Oral defence (viva voce): 04th August 2010 This submission was accepted on 20th July 2010 by the Faculty for Natural Sciences at the Universität Hohenheim as „Thesis presented in fulfilment of the thesis requirement for the degree of Doctor of Philosophy in Natural Sciences (Dr. rer. nat.)”. If I have seen further than others, it is by standing upon the shoulders of giants. - Isaac Newton This piece of work is dedicated to Ms. Hausmann, my former biology tutor. I sincerely thank her for inspiring me! Index 1 Introduction ............................................................................................................... 1 2 Material and methods ................................................................................................ 7 2.1 Available gravity research platforms ........................................................................... 7 2.2 Parabolic flight ............................................................................................................. 9 2.2.1 The aircraft ........................................................................................................... 9 2.2.2 The facilities ........................................................................................................ 10 2.3 The parabolic flight campaign ................................................................................... 11 2.3.1 The flight days .................................................................................................... 13 2.4 The parabolic flight manoeuvre ................................................................................ 15 2.4.1 Aircraft dynamics during the flight .................................................................... 16 2.4.2 Forces during the parabolic flight manoeuvre on the participants ................... 18 2.5 Classic patch clamp technique................................................................................... 22 2.5.1 Basic principle ..................................................................................................... 22 2.5.2 Supporting systems ............................................................................................ 23 2.5.3 The microscope .................................................................................................. 26 2.5.4 The patch clamp amplifier .................................................................................. 26 2.5.5 The micromanipulator ........................................................................................ 27 2.5.6 Electrodes ........................................................................................................... 27 2.5.7 Patch pipettes ..................................................................................................... 28 2.5.8 Pipette holder ..................................................................................................... 29 2.6 Patch clamp configurations ....................................................................................... 29 2.6.1 Whole cell recording .......................................................................................... 30 2.6.2 Single channel recording .................................................................................... 30 2.7 Practical execution..................................................................................................... 32 2.8 Planar patch clamp technique ................................................................................... 32 2.8.1 Basic principle ..................................................................................................... 33 I Index 2.8.2 The hardware ..................................................................................................... 33 2.8.3 The NPC-1 chip ................................................................................................... 34 2.8.4 Cell culture.......................................................................................................... 34 2.8.5 Patch clamp solutions ........................................................................................ 35 2.8.6 Practical execution ............................................................................................. 36 2.9 Cell culture ................................................................................................................. 36 2.9.1 Primary culture of human muscle cells .............................................................. 36 2.9.2 SH-SY5Y cell line ................................................................................................. 37 2.9.3 SNB19 cell line .................................................................................................... 37 2.9.4 Cultivation .......................................................................................................... 38 2.9.5 Splitting ............................................................................................................... 38 2.9.6 Differentiation .................................................................................................... 39 2.9.7 Viability check ..................................................................................................... 39 2.9.8 Cell harvesting for planar patch clamp .............................................................. 40 2.10 Adaptations for microgravity research ...................................................................... 40 2.10.1 Planar patch clamp pulse protocols ................................................................... 40 2.10.2 Cell culture.......................................................................................................... 41 2.10.3 Equipment .......................................................................................................... 41 2.10.4 Cultivation .......................................................................................................... 42 2.10.5 Time schedule..................................................................................................... 43 2.10.6 Storage during flight ........................................................................................... 43 2.11 Used software ............................................................................................................ 43 3 Results ..................................................................................................................... 44 3.1 Design of a planar patch clamp setup for microgravity research ............................. 45 3.1.1 Mechanical structure ......................................................................................... 45 3.1.2 Calculation of the structural properties ............................................................. 52 II Index 3.1.3 Components ....................................................................................................... 55 3.2 Sensory data .............................................................................................................. 68 3.2.1 Temperature ....................................................................................................... 68 3.2.2 Pressure .............................................................................................................. 69 3.2.3 Acceleration ........................................................................................................ 69 3.2.4 Magnetic field ..................................................................................................... 71 3.2.5 Power supply ...................................................................................................... 72 3.3 Patch clamp experiments under different gravity conditions ................................... 79 3.3.1 Current-voltage characteristics of undifferentiated SH-SY5Y cells .................... 79 3.3.2 Current-voltage characteristics of SNB19 cells .................................................. 84 3.3.3 Constant voltage clamp protocols of SNB19 cells .............................................. 86 4 Discussion and outlook ............................................................................................ 88 5 Abstract ................................................................................................................... 94 5.1 English abstract .......................................................................................................... 94 5.2 Deutsche Zusammenfassung ..................................................................................... 96 6 List of used abbreviations......................................................................................... 98 7 Table of figures ....................................................................................................... 100 8 References .............................................................................................................