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.

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• Publié par: universitat_zu_koln
• Publié le: 01 janvier 2009
• Nombre de lectures: 32
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

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Aus dem  Zentrum Physiolologie und Pathophysiologie der Universität zu Köln Institut für Neurophysiologie Geschäftsführender Direktor: Universitätsprofessor Dr. med. J. H

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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 Köln           vorgelegt von Abdulrhman Ahmed Akasha Aus Tripolis, Libyen     Promoviert am: 15. Juli 2009

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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.

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Bie der Auswahl und Auswertung des Materials sowie bei Herstellung des Manuskriptes

habe ich keine Unterstützungsleistungen erhalten.

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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.

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Die Arbeit wurde von mir bisher weder in Inland noch Ausland gleicher oder ähnlicher

Form einer anderen Prüfungsbehörde vorgelegt.

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Köln, den 9. Jan 2009

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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.

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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.

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I would like to acknowledge and thank Prof. Dr. Jürgen Hescheler, Director of Institute

of Neurophysiology, Cologne, for providing facilities for this study.

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I would like to thank all members of my group for their support, especially to:

Rita Altenburg, Michael Xavier Doss and Sania Sotiriadou.

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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.

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I would like to thank Dr. Johannes Winkler, for reading parts of my thesis.

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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.

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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.

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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.

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Table of contents Page   List of abbreviations 1

List of tables 2

List of figures 2 

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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

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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

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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

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3. Methods 29

 2.4 Equipment 28

 2.3 Plastic and glass materials 27

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 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

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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

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 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 + ytocesrdcamyio

 in CultiSphere-S by using Techne flask spinner 34

 2.2 materials 27

 2.1 Cell line 27

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2. Materials and Equipments 27

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  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

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 5.1 Isolation of highly purified α -MHC + cardiomyocytes from  a transgenic α - MHC +  55embryonic stem cell line  5.2 In vitro expansion of α -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

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uture work 60

  7. Summary in English and German 61

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 8. References 63

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Preliminary Publication 73

10. Curriculum Vitae 74

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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

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Angiotensin converting enzyme

EGFP

EB

DMEM

DMSO

FESEM

GFP

ES cell

ESCA

Embryonic stem cells aggregate Field emission scanning electron microscopy Green 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

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RA

NGF

MLC-2v

PBS

PA

List of Tables Page Table 1  24physical properties of CultiSpher-S & G microspheresThe

CultiSpher-S microsph

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Determination the average number of cells per CultiSpher-S bead Correlation between the number of cardiomyocytes

Table 2 Table 3

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SA

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eres

SEM

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Room temperature

Sinoatrial

Scanning electron microscope

Fig.12Diagrammatic illustration of Confocal laser scanning microscopy

 (CLSM) 38

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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

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List of figures

Average percentage of long-term entrapment of cardiomyocyte

and entrapment

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Table 4

  Fig. 13 Progressive generation of"-MHC + cardiomyocyte aggregates

 after treatment of the 8-day old (Ebs) with 4 µg/ml puromycin

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3

 for 6-7 days 39

Fig. 14 of attached and spreading ES-embryoid bodies Control

 without puromycin treatment 39 Fig. 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. 21eglrdcainSwseereymoitycotedfromdissociaeh-rSCluitpS

 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