Entrapment of {α-MHC [alpha-MHC] cardiomyocytes in CultiSpher-S microcarriers [Elektronische Ressource] : preparation and characterization / vorgelegt von Abdulrhman Ahmed Akasha

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Aus dem Zentrum Physiolologie und Pathophysiologie der Universität zu Köln Institut für Neurophysiologie Geschäftsführender Direktor: Universitätsprofessor Dr. med. J. Hescheler Entrapment of α-MHC Cardiomyocytes in CultiSpher-S Microcarriers: Preparation and Characterization Inaugural-Dissertation zur Erlangung der Würde eines doctor rerum medicinalium der Hohen Medizinischen Fakultät der Universität zu Köln vorgelegt von Abdulrhman Ahmed Akasha Aus Tripolis, Libyen Promoviert am: 15. Juli 2009 Dekanin/Dekan: Universitätsprofessor Dr. med. J. Klosterkötter 1. Berichterstatterin/ Berichterstatter: Professor Dr. rer. nat. A. Sachinidis 2. Berichterstatterin/ Btter: Frau Universitätsprofessor Dr. med. G. Pfitzer Erklärung Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als angegebenen Hilfsmittel angefertigt habe; die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche kenntlich gemacht. Bie der Auswahl und Auswertung des Materials sowie bei Herstellung des Manuskriptes habe ich keine Unterstützungsleistungen erhalten.
Publié le : jeudi 1 janvier 2009
Lecture(s) : 30
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Source : NBN-RESOLVING.DE/URN:NBN:DE:HBZ:38M-0000000871
Nombre de pages : 84
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Aus demZentrum Physiolologie und Pathophysiologie der Universität zu Köln Institut für Neurophysiologie Geschäftsführender Direktor: Universitätsprofessor Dr. med. J. H
escheler
Entrapment ofα-MHC Cardiomyocytes in CultiSpher-S Microcarriers: Preparation and Characterization Inaugural-Dissertation zur Erlangung der Würde eines doctor rerum medicinalium der Hohen Medizinischen Fakultät
 der Universität zu Kölnvorgelegt von Abdulrhman Ahmed Akasha Aus Tripolis, Libyen Promoviert am: 15. Juli 2009
Dekanin/Dekan:  Universitätsprofessor Dr. med. J. Klosterkötter 1. Berichterstatterin/ Berichterstatter:Professor Dr. rer. nat. A. Sachinidis 2. Berichterstatterin/ Berichterstatter:Frau Universitätsprofessor Dr. med. G.Pfitzer Erklärung Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und
ohne Benutzung anderer als angegebenen Hilfsmittel angefertigt habe; die aus fremden
Quellen direkt oder indirekt übernommenen Gedanken sind als solche kenntlich gemacht.
Bie der Auswahl und Auswertung des Materials sowie bei Herstellung des Manuskriptes
habe ich keine Unterstützungsleistungen erhalten.
Weitere Personen waren an der geistigen Herstellung der vorliegenden Arbeit nicht
beteiligt. Insbesondere habe ich nicht Hilfe eines Promotionsberaters in Anspruch
genommen. Dritte haben von mir weder unmittelbar noch mittelbar geldwerte Leistungen
für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation
stehen.
Die Arbeit wurde von mir bisher weder in Inland noch Ausland gleicher oder ähnlicher
Form einer anderen Prüfungsbehörde vorgelegt.
Köln, den 9. Jan 2009
Die in dieser Arbeit angegebenen Experimente sind nach entsprechender Anleitung
durch Herrn Prof. Dr. rer. nat. A. Sachinidis von mir mit Unterstützung von der
medizinisch-technichen Assistentin Frau Rita Altenburg durchgeführt worden.
Acknowledgment I wish to express my sincere thanks, gratitude and appreciation to my supervisor
Professor Dr. Agapios Sachinidis for his continuous help in overcoming difficulties
encountered in the course of study and for providing the right advice at the right time,
and for his encouragement, Advice and direction throughout all stages of this work.
I would like to acknowledge and thank Prof. Dr. Jürgen Hescheler, Director of Institute
of Neurophysiology, Cologne, for providing facilities for this study.
I would like to thank all members of my group for their support, especially to:
Rita Altenburg, Michael Xavier Doss and Sania Sotiriadou.
Additionally, cordial thanks to all members of the Institute of Neurophysiology for their
friendship, help and discussion over the years I spent in this institute.
I would like to thank Dr. Johannes Winkler, for reading parts of my thesis.
Many specific tasks involved in this study were performed in cooperation with other
research groups. For this work thanks go to:
Prof. Dr. Heiko Zimmermann and Jennifer Baunach, Fraunhofer Institut, St. Ingbert, for
their help to achieve the LSM and SEM investigations.
Dr. Marcel Halbach, Institute of Neurophysiology, Cologne, for his help to achieve the
Electrophysiological characterizations.
I would like to express my deep thanks to the Libyan cultural office/Libyan Embassy, for
their help, kindness and encouragement throughout the period of my study.
Extra-special thanks go to my wife (Somaia) for her enormous encouragement. She
endured tremendous hardship to allow me to complete this work. I would like to dedicate
this work to her and my Children.
This work was supported by EU grant (LSHB-CT-2006-03761) and is gratefully
acknowledged.
Table of contents PageList of abbreviations 1
List of tables 2
List of figures 2
1. Introduction 4
1.1 Heart 4
 1.1.1 Heart failure 7
 1.1.2 Symptoms of heart failure 8
 1.1.3 Causes of heart failure 8
 1.1.4 Treatment of heart failure 10
1.2 Stem cells 11
 1.2.1 Embryonic stem cells 11
 1.2.2 Characteristic of embryonic stem cells 13
 1.2.3 Potential benefits of ES cells in science and medicine 14
 1.2.4 Differentiation of Embryonic Stem cells
 into Cardiomyocytes 15
 1.2.5 Stem cells treatment of heart disease 17
1.3 Microcarriers 18
 1.3.1 microspheres 19
 1.3.2 Selection of matrix materials 20
 1.3.3 Applications of microcarriers in cell culture 22
 1.3.4 CultiSpher 23
 1.3.4.1 Scientific uses of CultiSpher-S 25
3. Methods 29
 2.4 Equipment 28
 2.3 Plastic and glass materials 27
 3.2 Preparation of emberyonic bodies and generation of ES-derivedα-MHC+Cardiomyocytes 29  3.3 Dissociation ofα-MHC+cardiomyocytes 31
 3.1 ES cell culture 29
4. Results 39
 4.1 Generation of 14 days old EGFP+" 39-MHC cardiomyocytes aggregates  4.2 Dissociation of GFP+" 40-MHC cardiomyocytes (without agitation)
 3.8 Long term growth and estimation of the entrapment efficiency  ofα-MHC+ 35cardiomyocytes in CultiSpher-S microspheres  3.9 Sharp electrode electrophysiological measurments 36
3.10 Block face scanning electron microscopy (SEM) of CultiSpher-S
 beads colonised by cardiomyocytes 36
3.11 Confocal laser scanning microscopy (CLSM) 38
 on CultiSphere-S carriers by GFL shaker 31 3.6 In vitro expansion ofα-MHC+craidmoyocytes
 3.7 Estimation of entrapped cardiomyocytes number per bead 35
 3.4 Preparation of hydrated and sterile CultiSpher-S mircrocarriers 31 3.5 In vitro expansion ofα-MHC+ytocesrdcamyio
 in CultiSphere-S by using Techne flask spinner 34
 2.2 materials 27
 2.1 Cell line 27
2. Materials and Equipments 27
 Page
 Page
 4.3 Control entrapment process without agitation 40 4.4 Entrapment (expansion) of GFP+"-MHC cardiomyocytes in CultiSpher-S by using shaker 41 4.5 Entrapment and spreading of GFP+"-MHC cardiomyocytes
 in a stirred falsk culture 42 4.6 Optimization of GFP+"-MHC cardiomyocytes entrapment
 in CultiSpher-S microspheres 43
4.7 Control entrapment of arterial cells derived from neonatal mouse
 heart on cultispher-S microspheres 43
 4.8 Dissociated cardiomyocytes from CultiSpher-S microspheres 44
4.9 Overview ofα-MHC cardiomyocytes entrapment
 in CultiSpher-S microspheres 44
4.10 Long-term growth of cardiomyocytes entrapped on cultispher-S
 microspheres 46
 4.11 Electrophysiological characterization of cardiomyocytes entrapped in
 CultiSpher-S microspheres 47
 4.12 Characterization of entrapped cells in CultiSpher-S by using
 confocal laser scanning microscope (LSM) 48
 4.13 Characterization of cells entrapped in CultiSpher-S by using
 scanning electron microscope (SEM) 52
 5. Discussion 55
 5.1 Isolation of highly purifiedα-MHC+cardiomyocytes from  a transgenicα- MHC+ 55embryonic stem cell line  5.2 In vitro expansion ofα-MHC+esytaccoymoidr  in CultiSpher-S microspheres 55
 5.3 Characterization of endogenous and exogenous expansion
 of cardiomycytes on CultiSpher-S microspheres 58
 5.4 Electrophysiological characterization of the
 entrapped cardiomyocytes 59
 Page
 6. F
uture work 60
 7. Summary in English and German 61
 8. References 63
9.
Preliminary Publication 73
10. Curriculum Vitae 74
CHF
CGR8
cDNA
Ca-Mg
AP
bFGF
BSE
CLSM
Backscattered electron
Calcium-Magnesium
Retinoic acid
Respiratory tract
BM-MSC
Action potential
basic fibroblast growth factor
Bone marrow mesenchymal stem cells
LIF
ACE
HepZ
hES cell
mES cell
MHC
List of abbreviations
Angiotensin converting enzyme
EGFP
EB
DMEM
DMSO
FESEM
GFP
ES cell
ESCA
Embryonic stem cells aggregate Field emission scanning electron microscopyGreen fluorescent protein
Polylactic-co-glycolic acid
Leukemia inhibitory factor
Human embryonic stem cell
Phosphate buffered saline
Hepatocyte cell line
Dimethylsulfoxide
Dulbecco modified Eagles minimal essential medium
Confocal laser scanning microscopy
Congestive heart failure
Mouse embryonic stem cell line CGR8
Complementary DNA
Polylactic acid
1
Polyanhydride
Nerve growth factor
Myosine light chain-2v
Myosine heavy chain
murine ES cell
Embryonic stem cell
Enhanced green fluorescent protein
Embryoid body
RT
PLGA
PLA
RA
NGF
MLC-2v
PBS
PA
List of Tables Page Table 1 24physical properties of CultiSpher-S & G microspheresThe
CultiSpher-S microsph
Determination the average number of cells per CultiSpher-S bead Correlation between the number of cardiomyocytes
Table 2 Table 3
45
RT
SA
45
eres
SEM
46
Room temperature
Sinoatrial
Scanning electron microscope
Fig.12Diagrammatic illustration of Confocal laser scanning microscopy
 (CLSM) 38
2
Fig. 7Steps of the protocol used for generating ES-derived "-MHC+cardi omyocytes 30
Fig. 8used for generating ES-cell derived cardiomyocytesProtocol  32 Fig. 9Progressive entrapment ofα-MHC+cardiomyocytes in CultiSpher-S 33
Fig. 10 34Diagrammatic illustration of Techne spinner flask
Fig. 11illustration of scanning electron microscopy (SEM) Diagrammatic  37
Fig. 4Differentiation of the human embryo into the three germ layers 12
Fig. 3Schematic diagram of the human heart 6
Fig. 6CultiSpher-S microspheres 25
Fig. 5 13Basic mechanisms of the heart development
 types of valves 5
Fig. 1illustration of the heart showing the four differentCross-section
 the atrial and ventricular chambers 5
Fig. 2The (SA) node and (AV) node of the heart, showing also
List of figures
Average percentage of long-term entrapment of cardiomyocyte
and entrapment
Table 4
Fig. 13 Progressive generation of"-MHC+cardiomyocyte aggregates
 after treatment of the 8-day old (Ebs) with 4 µg/ml puromycin
3
 for 6-7 days 39
Fig. 14 of attached and spreading ES-embryoid bodies Control
 without puromycin treatment 39Fig. 15 The 14 days old"-MHC+cardiomyocyte single_cell prior to agitation 40
Fig. 16Cardiomyocytes suspension cultures and CultiSpher-S microspheres
 Cultured without agitation for four day 40 Fig. 17 Progressive entrapment ofα-MHC+cardiomyocytes in CultiSpher-S  microspheres (day 7) by using small bioreactor 41 Fig. 18 Progressive entrapment ofα-MHC+cardiomyocytes in CultiSpher-S
 microspheres (day 7) by using Techne spinner flask 42 Fig. 19Optimum entrapment conditions for"-MHC+semyioytocrdca
 results in 80% of colonised CultiSpher-S microspheres 43
Fig. 20 Successful entrapment of arterial cells in CultiSpher-S beads 44
Fig. 21eglrdcainSwseereymoitycotedfromdissociaeh-rSCluitpS
 microspheres after entrapment progress (day7-21) 44
Fig. 22 Correlation between number of CultiSpher-S microsphers
 and entrapped cardiomyocytes 45 Fig. 23 Cell entrapment curve ofα-MHC+cardiomyocytes in CultiSpher-S  microcarriers 47 Fig. 24 Electrophysiological characterization ofα-MHC+cardiomyocytes on
 CultiSpher-S microspheres 48 Fig. 25 CLSM pictures of an entrapped CultiSpher-S microspheres withα-MHC+
cardiomyocytes (7 days old), of batch-1 49 Fig. 26 CLSM pictures of an entrapped CultiSpher-S microspheres withα-MHC+
cardiomyocytes (7 days old), of batch-2 50
Fig. 27 CLSM pictures of an entrapped CultiSpher-S microspheres with MHC+cardiomyocytes (7 days old), batch-3 51 α-Fig. 28 Block-face SEM inverted images of the CultiSpher-S microspheres  colonised withα-MHC+cardiomyocytes 54
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