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Neural Precursor Cells in Culture: Taking a Closer LookBernas, Stefanie 19 January 2019 (has links)
Gene mit gerigem Einfluss auf einen untersuchten Phänotyp können durch den Ein- schluss einer genetischen Variation im Tierversuch untersucht werden. Adulte Neuro- genese, der Prozess der Neubildung und Integration von funktionellen Neuronen in das existierende neurale Netzwerk, wird von vielen solchen Genen mit geringem Effekt beeinflusst. All diese Gene im lebenden Tier zu untersuchen wäre mit einem hohen Arbeitsaufwand verbunden, und würde hohe Tierzahlen erfordern. Bereits publizierte Ergebnisse zeigen, dass diese Gene auch in der Zellkultur unter Verwendung von Zelllinien genetisch rekombinanter Tiere untersucht werden können (Kannan et al., 2016). Die hier verwendeten, ingezüchteten Mausstämme des so genannten BXD Panels stellen die Nachkommen der Kreuzung der beiden Mausstämme C57BL/6J und DBA/2J dar (Peirce et al., 2004), die sich in der Ausprägung von unterschiedlichen Neurogenese bezogenen Phänotypen bereits deutlich unterscheiden (Kempermann et al., 2006). Durch die Verwendung der BXD Tiere wird hierbei die Aussagekraft der genetischen Variation mit dem Zellkultursystem verbunden. Die Aussagekraft dieser Studie ist jedoch darin limitiert, dass aufgrund des verwendeten Protokolls nur eine Zelllinie pro Mausstamm generiert werden konnte. Daher präsentiere ich hier ein neues Protokoll welches es erlaubt eine Zelllinie aus nur einem einzelnen Tier zu generieren. Diese Methode kombiniert zwei bestehende Zellkultursysteme, die Neurosphärenkultur und die Monolayerkultur. Es stellte sich heraus, dass die Überlebensrate der einzelnen Zelllinien vom biologischen Hintergrund der Zellen beeinflusst wird. So ist die Überlebensrate von Zellen der DBA/2J Mäuse deutlich schlechter als die der C57BL/6J oder die der F1 Generation aus der Verpaarung der beiden Stämme. Es zeigte sich allerdings, dass diese Überlebensrate nicht ausschließlich von der vorhandenen Anzahl proliferierender Zellen abhängt, da B6D2F1 (F1 Generation mit einem C57BL/6J Muttertier) signifikant weniger proliferierende (Ki67 positive) Zellen in vivo aufweisen, jedoch keine geringere Überlebensrate der Zelllinien haben. Eine hoch standardisierte, umfangreiche Analyse der Zelllinien aller vier Mausstämme (C57BL/6J, DBA/2J, und die zwei reziproken F1 Nachkommen BDF1 und DBF1) zeigte eine hohe Varianz innerhalb genetisch identischer Linien, was die Be- stimmung eines Effektes, der durch den genetischen Hintergrund der Linien verursacht wird, beeinträchtigte. Die Zelllinien werden signifikant von äußeren Faktoren beeinflusst, wie z.B. durch das Einfrieren der Zellen. Dies gibt Hinweise darauf, dass Untersuchungen in der Zellkultur genau geplant, kritisch hinterfragt, sowie möglichst alle potentiellen Einflussfaktoren gleich gehalten werden müssen. Nur so können valide, aussagekräftige Ergebnisse mit der Zellkultur gewonnen werden. Automatische Zellkultursysteme, neue Mikroskopieverfahren, sowie besser definierte Langzeitstudien werden unser Verständnis von Zellen in der Zellkultur deutlich verbessern und dabei ihren Wert, sowie bestehende Limitationen, endgültig klären.:List of Figures I
List of Tables II
List of Abbreviations III
List of Publications V
1. Introduction 1
1.1 Genetic variation in animal research 2
Recombinant inbred strains 3
The BXD panel 4
The Gene Network 5
Genetic modifications 5
1.2 Adult hippocampal neurogenesis 6
History 7
Clinical relevance 8
The BXD panel and adult hippocampal neurogenesis 9
1.3 Developmental stages of neural precursor cells 9
1.4 Studying adult neurogenesis in vitro 11
Culturing hippocampal precursor cells 11
A mouse cell culture genetic reference panel 13
1.5 Tracking 13
1.6 Objectives 15
2. Materials and Methods 16
2.1 Components and equipment 16
2.2 Antibodies 20
2.3 Recipes 21
General buffers and solutions 21
Cell culture solutions 21
Immunocytochemistry solutions 23
Immunohistochemistry solutions 24
2.4 Experimental animals 25
2.5 Cell culture 25
Coating of cell culture vessels 25
Fire-polished pipettes 25
Dentate gyrus isolation 26
Neurosphere assay 26
Monolayer culture 27
2.6 Immunocytochemistry 29
BrdU staining preparations 29
Staining protocol 30
Imaging and counting 30
2.7 Immunohistochemistry 30
Sample preparation 30
Staining protocol 31
Cell counting 31
2.8 Tracking 32
Cell preparation and imaging setup 34
Image processing 35
Data analysis 35
2.9 Generation of CRISPR/Cas mediated knock-out lines 36
Construct design and cloning 36
E. coli Top10 transformation and plasmid isolation 37
Transfection of neural precursor cells and expansion of knock-out lines 38
Genotyping of the generated cell lines 39
Agarose gel electrophoresis 40
2.10 Statistical analysis 40
2.11 Data visualization 40
3. Results 41
3.1 Single animal monolayer cultures41
The three phenotypes of the neurosphere assay 44
Neurosphere assay phenotypes could not predict the survival of a cell line 45
The genetic background had an influence on all three phenotypes of the neurosphere assay 46
Significantly less proliferating cells in vivo but no difference in the neurosphere assay of BDF1 compared to BL6 animals 48
BDF1 cells could not be activated to form more spheres but sphere size could be increased using KCl 49
3.2 A new cell line phenotyping standard operation procedure and its application 50
Line generation data 52
Marker staining 54
Cell tracking 55
3.3 Cell culture – a system with limitations 59
Freezing effect 60
Cell culture data - technical variance hinders the analysis of small effects 62
3.4 Migration speed and GFAP 63
The strength of the BXD panel – cumulative data 65
3.5 Other applications of the tracking procedure 68
Tracking labeled cells in an embryonic zebrafish xenograft model 68
Cell tracking in mouse retina explants 68
4. Discussion 70
4.1 Single animal monolayer cultures – a new protocol 70
4.2 A new phenotyping pipeline 74
4.3 Semi-automated (user-supervised) cell tracking 77
4.4 A possible correlation between migration speed and differentiation 79
4.5 CRISPR/Cas knock-out lines - an ill-conceived system with high potential 82
4.6 The problem of the validity of cell culture experiments - a comment 83
4.7 Conclusion 84
Bibliography 88
A Single animal cell line generation protocol 106
B Cell line characterization SOP 112
C R Scripts 117 / Uncovering gene loci that assert only small effects onto a phenotype of interest, can be achieved by including genetic variation in animal research. Adult hippocampal neurogenesis, the process of the formation of new neurons and their functional integration into existing circuitry, is influenced by a broad range of such small effect genes. Analyzing all of these genes in vivo would be laborious and require a high number of animals. Previously published data merged the power of genetic variation with a cell culture system by using cell lines generated from the BXD recombinant inbred mouse strains (Kannan et al., 2016). These strains are inbred progeny of F2 crosses originating from the two mouse strains C57BL/6J and DBA/2J (Peirce et al., 2004), which already differ quite extensively in neurogenesis related phenotypes (Kempermann et al., 2006). As previous studies were limited by the number of strains that could be generated due to the demand for high numbers of animals, I developed a new method that allows the generation of a cell line from one single animal. For this new method, I combined the neurosphere culture with a subsequent monolayer culture. The survival of the resulting cell lines, is thereby greatly influenced by the genetic background. The survival rate of cell lines derived from DBA/2J animals is much lower as compared to C57BL/6J-derived lines or lines from the F1 generation of crossing the two strains. Whether or not a cell line survived did not seem to be solely influenced by the number of proliferating cells in vivo, as B6D2F1 (F1 progeny with a C57BL/6J mother) showed significantly less proliferative (Ki67 positive) cells in vivo while exhibiting a survival rate that exceeded both parental strains. An extensive study of the cell lines gained from all four mouse strains (C57BL/6J, DBA/2J, and the two reciprocal F1 progeny B6D2F1 and D2B6F1) in a highly standardized manner showed that the individual difference between single cell lines was rather high, hampering the successful detection of in-between strain differences. The standardized characterization of the generated cell lines, further allowed the identification of external factors, influencing the cells, as for example the freezing of the cells. This indicates that cell culture experiments need to be thoroughly planned and critically scrutinized, while all external factors should be kept as constant as possible to ensure the validity of the resulting data. Automated cell handling, new imaging technologies, as well as more defined long-term studies will greatly improve the understanding of cells in culture and thereby show their true values and limitations.:List of Figures I
List of Tables II
List of Abbreviations III
List of Publications V
1. Introduction 1
1.1 Genetic variation in animal research 2
Recombinant inbred strains 3
The BXD panel 4
The Gene Network 5
Genetic modifications 5
1.2 Adult hippocampal neurogenesis 6
History 7
Clinical relevance 8
The BXD panel and adult hippocampal neurogenesis 9
1.3 Developmental stages of neural precursor cells 9
1.4 Studying adult neurogenesis in vitro 11
Culturing hippocampal precursor cells 11
A mouse cell culture genetic reference panel 13
1.5 Tracking 13
1.6 Objectives 15
2. Materials and Methods 16
2.1 Components and equipment 16
2.2 Antibodies 20
2.3 Recipes 21
General buffers and solutions 21
Cell culture solutions 21
Immunocytochemistry solutions 23
Immunohistochemistry solutions 24
2.4 Experimental animals 25
2.5 Cell culture 25
Coating of cell culture vessels 25
Fire-polished pipettes 25
Dentate gyrus isolation 26
Neurosphere assay 26
Monolayer culture 27
2.6 Immunocytochemistry 29
BrdU staining preparations 29
Staining protocol 30
Imaging and counting 30
2.7 Immunohistochemistry 30
Sample preparation 30
Staining protocol 31
Cell counting 31
2.8 Tracking 32
Cell preparation and imaging setup 34
Image processing 35
Data analysis 35
2.9 Generation of CRISPR/Cas mediated knock-out lines 36
Construct design and cloning 36
E. coli Top10 transformation and plasmid isolation 37
Transfection of neural precursor cells and expansion of knock-out lines 38
Genotyping of the generated cell lines 39
Agarose gel electrophoresis 40
2.10 Statistical analysis 40
2.11 Data visualization 40
3. Results 41
3.1 Single animal monolayer cultures41
The three phenotypes of the neurosphere assay 44
Neurosphere assay phenotypes could not predict the survival of a cell line 45
The genetic background had an influence on all three phenotypes of the neurosphere assay 46
Significantly less proliferating cells in vivo but no difference in the neurosphere assay of BDF1 compared to BL6 animals 48
BDF1 cells could not be activated to form more spheres but sphere size could be increased using KCl 49
3.2 A new cell line phenotyping standard operation procedure and its application 50
Line generation data 52
Marker staining 54
Cell tracking 55
3.3 Cell culture – a system with limitations 59
Freezing effect 60
Cell culture data - technical variance hinders the analysis of small effects 62
3.4 Migration speed and GFAP 63
The strength of the BXD panel – cumulative data 65
3.5 Other applications of the tracking procedure 68
Tracking labeled cells in an embryonic zebrafish xenograft model 68
Cell tracking in mouse retina explants 68
4. Discussion 70
4.1 Single animal monolayer cultures – a new protocol 70
4.2 A new phenotyping pipeline 74
4.3 Semi-automated (user-supervised) cell tracking 77
4.4 A possible correlation between migration speed and differentiation 79
4.5 CRISPR/Cas knock-out lines - an ill-conceived system with high potential 82
4.6 The problem of the validity of cell culture experiments - a comment 83
4.7 Conclusion 84
Bibliography 88
A Single animal cell line generation protocol 106
B Cell line characterization SOP 112
C R Scripts 117
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