Mesenchymal stromal cells for cell therapy: good manufacturing practice-compliant expansion systems and nanoparticle-based imaging
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | VIII, 138 S. Ill., graph. Darst. |
Internformat
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| adam_text | IMAGE 1
LIST OF CONTENTS
1 INTRODUCTION 1
1.1 DEFINING MESENCHYMAL STROMAL CELLS (MSCS) 1
1.2 IMMUNOMODULATORY AND REGENERATIVE PROPERTIES O F MSCS 2
1.3 CULTIVATED ADULT STEM CELLS AS ALTERNATIVE FOR DAMAGED TISSUE
(CASCADE CONSORTIUM) 4
1.4 REGULATORY REQUIREMENTS FOR CELL-BASED THERAPIES 4
1.5 GMP-COMPLIANT MSC PRODUCTION FOR CLINICAL APPLICATION 5
1.5.1 HUMAN PLATELET LYSATE AS CELL CULTURE MEDIA SUPPLEMENT 6
1.5.2 STATIC CELL CULTURE SYSTEMS 6
1.5.3 BIOREACTOR PROTOTYPE (QUANTUM CELL EXPANSION SYSTEM) 7
1.6 ESSENTIAL COMPONENTS FOR EX VIVO PROLIFERATION O F MSCS 7
1.7 TESTING NEW SURFACES FOR CULTIVATION O F BM-MSCS: HIGHLY ORDERED
PYROLYTIC GRAPHITE (HOPG) 8
1.8 APPLICATION OFMSCS IN VIVO 9
1.8.1 REGULATORY ASPECTS 9
1.8.2 CHARACTERIZATION OF MSC SURFACE RECEPTOR PROFILE 9
1.8.3 MSC MIGRATION IN VITRO 9
1.8.4 MONITORING NANOPARTICLE-LABELED MSCS IN VIVO VIA MAGNETIC
RESONANCE (MR) AND INFRARED (IR) IMAGING 10
1.8.5 BIODISTRIBUTION OFMSCS IN VIVO 10
1.8.6 SILICONE CARRIERS FOR TOPICAL APPLICATION OFMSCS 11
1.9 HIGH-DOSE RADIATION OFMSCS FOR CLINICAL APPLICATION 11
1.10 MSC SUBPOPULATIONS 12
1.11 SCOPE O F THIS WORK 13
2 MATERIALS A N D M E T H O D S 14
2.1 ISOLATION AND EXPANSION OFMSCS 14
2.1.1 BONE MARROW COLLECTION 14
2.1.2 PROTOCOLS FOR GMP-COMPLIANT LARGE-SCALE ISOLATION IN STATIC TISSUE
CULTURE 14 2.1.2.1 SINGLE-STEP PROTOCOL 14
2.1.2.2 TWO-STEP PROTOCOLS 15
2.1.3 DIFFERENTIATION AND SENESCENCE ASSAYS 17
2.1.3.1 HISTOCHEMISTRY 17
2.1.3.2 REAL-TIME REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION
(REAL-TIME RT-PCR) AND SEMI QUANTITATIVE PCR 17
2.1.4 MODIFIED CFU-F ASSAY 17
2.1.5 BACTERIAL AND MICROBIAL TESTINGS 18
2.2 QUANTUM CELL EXPANSION SYSTEM 18
2.2.1 SYSTEM DESCRIPTION 18
2.2.2 T-25 PARALLEL CULTURE IN STATIC TISSUE CULTURE 20
2.3 MSCS ISOLATION AND EXPANSION USING SERUM-FREE SPE-IV MEDIUM
(ABCELL-BIO) 20
2.4 FORMULAS AND CALCULATIONS 21
2.5 MEDIA AND SUPPLEMENTS 23
2.5.1 PRODUCTION OF PLATELET LYSATE FROM WHOLE BLOOD DERIVED POOLED
PLATELET CONCENTRATE (PL-PPC)...23 2.5.2 PRODUCTION OF PLATELET LYSATE
FROM APHERESIS PLATELET CONCENTRATE (PL-APC) 24 2.5.3 PRODUCTION OF
HUMAN AB SERUM (ABS) 25
2.5.4 ISOLATION AND EXPANSION OFMSCS USING FCS AS A SUPPLEMENT 25
2.5.5 COMPARISON OF CELL CULTURE MEDIA 25
III
HTTP://D-NB.INFO/1027580750
IMAGE 2
2.5.6 NEUTRALIZING ASSAY 25
2.5.7 FACTORS TESTED AND COMBINED AS SYNTHETIC SERUM-FREE MEDIUM 26
2.5.7.1 ADDITION OF HUMAN INSULIN, INSULIN-TRANSFERRIN-SELENIUM (ITS)
AND GLUCOSE TO PROMOTE MSC PROLIFERATION 26
2.5.8 CELL CULTURE SURFACE COATING 27
2.6 CELL PROLIFERATION ASSAY 27
2.7 ANALYSIS O F CYTOKINES AND METABOLITES 27
2.7.1 LUMINEX ANALYSIS OF MEDIA AND SUPPLEMENTS 28
2.7.2 LACTATE AND GLUCOSE ANALYSIS 28
2.8 FLOW CYTOMETRY 29
2.8.1 ANNEXIN V STAINING 30
2.8.2 SORTING OF MSC SUBPOPULATIONS 30
2.9 MSC MIGRATION IN VITRO 31
2.9.1 IN VITRO TRANSWELL MIGRATION ASSAY 31
2.9.2 ORIS CELL MIGRATION ASSAY 31
2.9.2.1 MSC STIMULATION 33
2.10 MAGNETIC RESONANCE IMAGING 33
2.10.1 MRI CHARACTERIZATION OF NANOPARTICLES 35
2.11 PREPARATION O F NANOPARTICLES 36
2.12 NANOPARTICLE-LABELING OFMSCS 36
2.13 MAGNETIC RESONANCE IMAGING (MRI) ANALYSES IN VITRO AND IN VIVO 37
2.13.1 MRI ANALYSIS OF RELAXIVITY PHANTOMS 37
2.13.2 MRI ANALYSIS OF SINGLE CELLS IN VITRO 37
2.13.3 M R IMAGING OF SILICONE CARRIERS IN VITRO 37
2.13.4 M R IMAGING OF SILICONE CARRIERS IN VIVO 38
2.14 ISOLATION AND CULTIVATION O F RAT BONE MARROW-DERIVED MSCS 38
2.15 DEFINED SILICONE CARRIERS FOR TOPICAL APPLICATION O F MSCS 38
2.15.1 PREPARATION O F PPAAC-COATED SILICONE CARRIERS 38
2.15.2 CULTIVATION O F MSCS ON SILICONE CARRIERS 39
2.16 ANIMAL MODEL 39
2.17 INFRARED FLUORESCENCE IMAGING 39
2.18 HISTOLOGY 40
2.19 MSC CULTIVATION ON H O P G 40
2.19.1 PREPARATION O F H O P G 40
2.19.2 MSC CULTIVATION AND PROLIFERATION ON H O P G 41
2.20 SCANNING ELECTRON MICROSCOPY (SEM) 41
2.21 IRRADIATION O F MSCS 41
2.21.1 REAL-TIME POLYMERASE-CHAIN REACTION (RT-PCR) 41
2.22 STATISTICS 41
3 RESULTS A N D DISCUSSION 42
3.1 CHARACTERIZATION O F PLATELET LYSATE 42
3.1.1 MAJOR COMPONENTS OF PL-PPC 43
3.1.2 STABILITY OF PL 44
3.1.3 GROWTH PROMOTING EFFECT OF PL-PPC AND PL-APC 44
3.1.4 GROWTH PROMOTING EFFECT OF PL-PPC COMPARED TO FCS 45
3.1.5 CHARACTERIZATION OF LARGE-SCALE EXPANDED MSCS USING PL 45
3.1.6 INFLUENCE OF PDGF-BB ON MSC PROLIFERATION 46
IV
IMAGE 3
3.1.7 INHIBITION O F PL-INDUCED MSC PROLIFERATION 47
3.2 CHARACTERIZATION O F MSCS ISOLATED AND EXPANDED ACCORDING TO
GMP-COMPLIANT PROTOCOLS IN STATIC TISSUE CULTURE 48
3.2.1 ANALYSIS OF STARTING MATERIAL 48
3.2.2 ANALYSIS OF MSC YIELD, POPULATION DOUBLINGS AND CLONOGENICITY 48
3.2.3 PHENOTYPIC CHARACTERIZATION OF MSCS BY FLOW CYTOMETRY 49
3.2.4 IN VITRO DIFFERENTIATION CAPACITY 53
3.2.5 ANALYSIS OF METABOLIC PARAMETERS AND CYTOKINE PROFILES IN THE
MEDIUM 54
3.2.6 CONCLUSIVE REMARKS ON MSCS PRODUCED ACCORDING TO GMP-COMPLIANT
LARGE-SCALE PROTOCOLS 55
3.3 CHARACTERIZATION O F MSCS ISOLATED AND EXPANDED IN THE QUANTUM
SYSTEM 57
3.3.1 MSCS ISOLATION AND EXPANSION IN THE QUANTUM USING FCS-SUPPLEMENTED
MEDIA 57 3.3.2 MSCS ISOLATION AND EXPANSION IN A XENOGEN-FREE QUANTUM
SYSTEM 57
3.3.3 TESTING ALTERNATIVE REAGENTS FOR BIOREACTOR COATING 57
3.3.4 EFFECT OF CRYOPRESERVATION ON MSC GROWTH IN THE QUANTUM SYSTEM 58
3.3.5 PHENOTYPICAL CHARACTERIZATION OF MSCS CULTIVATED IN THE QUANTUM
SYSTEM 58 3.3.6 DIFFERENTIATION POTENTIAL OF MSCS CULTIVATED IN THE
QUANTUM SYSTEM 59
3.3.7 EXTENDED SURFACE MARKER PHENOTYPE OF MSCS CULTIVATED IN THE
QUANTUM SYSTEM 60 3.3.8 QUANTIFICATION OF SOLUBLE CYTOKINES IN MSC
CULTURE MEDIUM IN THE BIOREACTOR 63 3.3.9 CONCLUSIVE REMARKS ON THE
ISOLATION AND EXPANSION OF MSCS IN THE QUANTUM SYSTEM 67
3.4 CHARACTERIZATION O F MSCS ISOLATED AND EXPANDED IN SPE-1V SERUM-FREE
MEDIUM 68
3.4.1 MSCS ISOLATION FROM BONE MARROW AFTER DENSITY GRADIENT
CENTRIFUGATION 68
3.4.2 ANALYSIS OF MINIMAL CRITERIA OF MSCS CULTIVATED IN SPE-IV MEDIUM
68
3.4.3 EXTENDED SURFACE RECEPTOR PROFILE OF MSCS CULTIVATED IN SPE-IV
MEDIUM 69 3.4.4 METABOLIC MARKERS AND CYTOKINE PROFILE OF MSCS
CULTIVATED IN SPE-IV MEDIUM 72 3.4.5 ISOLATION AND EXPANSION OF MSCS
FROM UNPROCESSED BM IN SPE-IV MEDIUM 73 3.4.6 CONCLUSIVE REMARKS 74
3.5 ESSENTIAL COMPONENTS FOR EX VIVO PROLIFERATION O F MSCS 75
3.5.1 MSCS ISOLATION AND EXPANSION IN MEDIA SUPPLEMENTED WITH FCS 75
3.5.2 EFFECT OF BASAL MEDIA AND SUPPLEMENT COMPOSITIONS ON MSC
PROLIFERATION 75 3.5.3 EFFECT OF CELL SEEDING DENSITY 76
3.5.4 HEAT-INACTIVATION OF HUMAN OR ANIMAL-DERIVED MEDIA SUPPLEMENTS 77
3.5.5 DEFINED SERUM-FREE MEDIUM 79
3.5.6 EFFECT OF AUXILIARY GLUCOSE 79
3.5.7 INFLUENCE OF LIPIDS 81
3.5.8 COATING OF CELL CULTURE SURFACES 81
3.5.9 CONCLUSION 83
3.6 ADHESION TO HIGHLY ORDERED PYROLYTIC GRAPHITE (HOPG) 84
3.7 MSC MIGRATION IN VITRO 88
3.7.1 CHARACTERIZATION OF CHEMOKINE RECEPTOR PROFILE 88
3.7.2 TRANSWELL MIGRATION 90
3.7.3 ORIS CELL MIGRATION ASSAY 91
3.8 NANOPARTICLE-BASED IMAGING OF MSCS 94
3.8.1 CHARACTERIZATION OF NANOPARTICLES 94
3.8.2 IN VITRO MRI ANALYSES 95
3.8.3 DETECTION OF A SINGLE NANOPARTICLE-LABELED MSCS VIA MRI 96
3.9 IMAGING O F NANOPARTICLE-LABELED MSCS AFTER INJECTION IN A RAT MODEL
96
3.9.1 M R AND IR IMAGING OF LABELED MSCS IN VIVO : SCENARIO 1 96
3.9.2 M R AND IR IMAGING OF LABELED MSCS IN VIVO : SCENARIO 2 98
3.10 IMAGING O F NANOPARTICLE-LABELED MSCS AFTER TOPICAL DELIVERY VIA
SILICONE CARRIERS 103
3.10.1 MSCS ADHESION TO DEFINED SILICONE CARRIERS 103
3.10.2 M R IMAGING OF NANOPARTICLE-LABELED MSCS ON PPAAC-COATED CARRIERS
IN VITRO 103
V
IMAGE 4
3.10.3 M R IMAGING O F NANOPARTICLE-LABELED MSCS ON PPAAC-COATED
CARRIERS IN VIVO 103
3.10.4 CONCLUSION 107
3.11 CHARACTERIZATION O F IRRADIATED BM-DERIVED MSCS 108
3.11.1 CELL PROLIFERATION 108
3.11.2 DIFFERENTIATION CAPACITY AND CELLULAR SENESCENCE OF IRRADIATED
CELLS 109
3.11.3 CYTOKINE ANALYSES I L L
3.11.4 CELL CYCLE, TELOMERASE ACTIVITY AND TRANSFORMATION MARKERS 114
3.12 CD349* AND MSCA-1* MSC SUBPOPULATIONS 116
4 CONCLUSIONS A N D OUTLOOK 118
4.1 GMP-COMPLIANT PROCEDURES FOR CLINICAL-SCALE PRODUCTION OF MSCS 118
4.1.1 MSC ISOLATION AND EXPANSION IN STATIC TISSUE CULTURE SYSTEMS 118
MSC ISOLATION AND EXPANSION IN THE QUANTUM SYSTEM 119
4.2 EFFECT O F CELL CULTURE MEDIA AND SUPPLEMENTS ON MSCS 119
4.2.1 PLATELET LYSATE AS CELL CULTURE MEDIA SUPPLEMENT 120
4.2.2 ESSENTIAL COMPONENTS FOR MSC PROLIFERATION 120
4.2.3 SERUM-FREE SPE-IV MEDIUM 121
4.3 INTERACTION O F MSCS AND CULTURE SURFACES 121
4.4 IMAGING OF MSCS IN VIVO 122
4.5 IRRADIATION O F MSCS 123
4.6 SORTING O F MSC SUBPOPULATIONS 124
5 SUMMARY 125
6 ZUSAMMENFASSUNG 127
7 REFERENCES 129
8 APPENDIX 133
8.1 ACKNOWLEDGEMENTS 133
8.2 PUBLICATIONS 134
8.3 CURRICULUM VITAE 137
8.4 EIDESSTATTLICHE ERKLARUNG 138
VI
|
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| spelling | Fekete, Natalie 1984- Verfasser (DE-588)1027561403 aut Mesenchymal stromal cells for cell therapy good manufacturing practice-compliant expansion systems and nanoparticle-based imaging vorgelegt von Natalie Fekete 2012 VIII, 138 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Ulm, Univ., Diss., 2012 Frischzellentherapie (DE-588)4067560-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Frischzellentherapie (DE-588)4067560-9 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025389564&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Fekete, Natalie 1984- Mesenchymal stromal cells for cell therapy good manufacturing practice-compliant expansion systems and nanoparticle-based imaging Frischzellentherapie (DE-588)4067560-9 gnd |
| subject_GND | (DE-588)4067560-9 (DE-588)4113937-9 |
| title | Mesenchymal stromal cells for cell therapy good manufacturing practice-compliant expansion systems and nanoparticle-based imaging |
| title_auth | Mesenchymal stromal cells for cell therapy good manufacturing practice-compliant expansion systems and nanoparticle-based imaging |
| title_exact_search | Mesenchymal stromal cells for cell therapy good manufacturing practice-compliant expansion systems and nanoparticle-based imaging |
| title_full | Mesenchymal stromal cells for cell therapy good manufacturing practice-compliant expansion systems and nanoparticle-based imaging vorgelegt von Natalie Fekete |
| title_fullStr | Mesenchymal stromal cells for cell therapy good manufacturing practice-compliant expansion systems and nanoparticle-based imaging vorgelegt von Natalie Fekete |
| title_full_unstemmed | Mesenchymal stromal cells for cell therapy good manufacturing practice-compliant expansion systems and nanoparticle-based imaging vorgelegt von Natalie Fekete |
| title_short | Mesenchymal stromal cells for cell therapy |
| title_sort | mesenchymal stromal cells for cell therapy good manufacturing practice compliant expansion systems and nanoparticle based imaging |
| title_sub | good manufacturing practice-compliant expansion systems and nanoparticle-based imaging |
| topic | Frischzellentherapie (DE-588)4067560-9 gnd |
| topic_facet | Frischzellentherapie Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025389564&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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