Analysis and modification of biosilica‐forming sponge (Suberites domuncula) primmorph cells and hydroxyapatite‐forming human SaOS‐2 cells Dissertation zur Erlangung des Grades "Doktor der Naturwissenschaften" am Fachbereich Biologie der Johannes Gutenberg‐Universität in Mainz vorgelegt von Julia Sofia Markl geb. am 02. Oktober 1985 in München Mainz, 2016 Dekan: Prof. Dr. Hans Zischler Erster Berichterstatter: Prof. Dr. Werner E. G. Müller Zweiter Berichterstatter: Prof Dr. Walter Stöcker Tag der mündlichen Prüfung: 27.06.2016 D77 (Dissertation Johannes Gutenberg‐Universität Mainz) INDEX 1 SUMMARY ......................................................................................................................................... 1 2 ZUSAMMENFASSUNG .......................................................................................................................... 2 3 INTRODUCTION .................................................................................................................................. 3 3.1 Sponges as members of the animal kingdom ......................................................................... 3 3.2 Sponge general morphology and systematics ........................................................................ 5 3.3 Biosilica production in sponges ............................................................................................... 8 3.4 The sponge Suberites domuncula in nature and captivity .................................................... 15 3.5 S. domuncula and its biosilica spicules in biological and material research ......................... 16 3.6 The primmorph: a sponge cell accumulation in culture ....................................................... 17 3.7 Phagocytosis in sponges and primmorphs, and application of nanoparticles ...................... 19 3.8 Apatite formation enhanced by silicon in human bone‐forming (SaOS‐2) cells ................... 20 3.9 Aims of the present study ..................................................................................................... 22 4 MATERIAL AND METHODS ................................................................................................................. 23 4.1 Material ................................................................................................................................. 23 4.1.1 Consumables ................................................................................................................. 23 4.1.2 Reagents ........................................................................................................................ 24 4.1.3 Buffers and solutions ..................................................................................................... 27 4.1.4 Kits ................................................................................................................................. 28 4.1.5 DNA plasmids ................................................................................................................ 29 4.1.6 Culture media ................................................................................................................ 29 4.1.7 Animals and cells ........................................................................................................... 30 4.1.8 Instruments ................................................................................................................... 31 4.1.9 Software ........................................................................................................................ 34 4.2 General procedures ............................................................................................................... 34 4.3 Assembly of magnetic nanoscale building blocks to sponge biosilica spicules ..................... 35 4.3.1 Isolation of sponge spicules .......................................................................................... 35 4.3.2 Silanization of spicules .................................................................................................. 35 4.3.3 EDC‐activation of nano‐screenMAG‐CMX nanoparticles and binding to spicules ........ 35 4.4 A bioreactor procedure to raise exceptionally large primmorphs ........................................ 37 4.4.1 Standard primmorph preparation ................................................................................. 37 4.4.2 Sponge cell culture ........................................................................................................ 39 4.4.3 How to use the custom made bioreactor ..................................................................... 39 4.5 Uptake of magnetic and fluorescent particles ...................................................................... 39 4.5.1 Incubation of primmorphs with nano‐ and microparticles ........................................... 39 4.5.2 Frozen tissue sectioning and fluorescence microscopy ................................................ 40 4.5.3 Chemical fixation and sectioning for electron microscopy ........................................... 40 4.5.4 Transmission electron microscopy and energy‐dispersive X‐ray (EDX) spectroscopy .. 41 4.5.5 Scanning electron microscopy ...................................................................................... 42 4.5.6 RNA preparation ............................................................................................................ 42 4.5.7 Agarose gel electrophoresis .......................................................................................... 42 4.5.8 cDNA preparation from RNA ......................................................................................... 43 4.5.9 Quantitative PCR (qPCR) ............................................................................................... 44 4.6 Apatite formation of SaOS‐2 cells transfected with sponge silicatein‐α .............................. 46 4.6.1 Gateway cloning of silicatein‐α in pcDNA6.2 cTC ......................................................... 46 4.6.2 Amplification and purification of plasmids from E. coli for transfection ...................... 51 4.6.3 SaOS‐2 cell culture ......................................................................................................... 52 4.6.3.1 Daily culturing of SaOS‐2 cells ................................................................................... 52 4.6.3.2 Activation of SaOS‐2 cells .......................................................................................... 53 4.6.3.3 Transfection and selection of SaOS‐2 cells ................................................................ 53 4.6.3.4 Cell fixation ................................................................................................................ 54 4.6.3.5 Alizarin red assay ....................................................................................................... 55 4.6.3.6 AlamarBlue assay ...................................................................................................... 55 4.6.3.7 Alkaline phosphatase activity assay and protein concentration assay ..................... 55 4.6.3.8 Staining of hydroxyapatite ........................................................................................ 56 4.6.3.9 Staining of cell nuclei ................................................................................................. 57 4.6.3.10 Staining of the actin cytoskeleton ......................................................................... 57 4.6.3.11 Complete staining procedure ................................................................................ 57 4.6.3.12 Statistical Analysis ................................................................................................. 58 4.7 Transcriptome analysis of sponge tissues adapted to daylight and night ............................ 58 4.7.1 Incubation of sponges ................................................................................................... 58 4.7.2 Transcriptome analysis .................................................................................................. 59 5 RESULTS .......................................................................................................................................... 60 5.1 Transcriptome and quantitative PCR analyses on mature sponges ...................................... 60 5.2 A bioreactor‐based procedure to improve sponge primmorph cell culture ......................... 62 5.3 Uptake of fluorescent and magnetic nanoparticles by primmorph cells .............................. 66 5.3.1 Experimental setting, and comparative tests with different types of nanoparticles ... 66 5.3.2 Demonstration of nanoparticle uptake by fluorescence microscopy and magnets ..... 67 5.3.3 Verification of nanoparticle uptake by TEM, TEM‐EDX and SEM‐EDX .......................... 70 5.3.4 Quantitative PCR (qPCR) of spiculogenesis‐specific RNAs after nanoparticle uptake .. 76 5.4 Layer‐by‐layer assembly of nanoscale building blocks to biosilica spicules ......................... 77 5.5 Uptake of fluorescent silica core‐shell microparticles .......................................................... 80 5.6 Influence of silicatein‐α expression on apatite formation by SaOS‐2 cells ........................... 84 6 DISCUSSION ..................................................................................................................................... 90 6.1 Transcriptome and qPCR analyses of sponge