Title Page
Contents
GENERAL INTRODUCTION 13
PART I. Functional recovery by colon organoid transplantation in a mouse model of radiation proctitis 16
Abstract 17
I. INTRODUCTION 19
II. MATERIALS AND METHODS 21
1. Animals 21
2. Model of Radiation-Induced Proctitis 21
3. Mouse Colon organoid Transplantation and Endoscopic Procedure 22
4. RT-qPCR Analysis 23
5. Regeneration Area Measurement in Engraftment Tissues 23
6. Tetramethylrhodamine (TRITC)-Dextran Permeability Assay 24
7. Immunohistochemistry for Engraftment Tissue Analysis 24
8. Single Cell RNA Sequencing Analysis of Colon organoid Engrafted Tissue 25
9. Colon organoid Cryopreservation and Transplantation 25
10. Statistical analysis 26
III. RESULTS 27
1. Optimization of a radiation proctitis mouse model for evaluating efficacy of colon organoids 27
2. Engraftment of transplanted colon organoids in radiation proctitis mouse model 33
3. Therapeutic effects of engrafted colon organoids in the radiation proctitis mouse model 39
4. Lgr5+ stem cell is an essential component for therapeutic effects of colon organoids[이미지참조] 56
5. Therapeutic approaches by endoscopic transplantation of colon organoids 64
IV. DISCUSSION 67
V. REFERENCES 70
ABSTRACT IN KOREAN 76
PART II. In vivo evaluation of scaffolds compatible for colonoid engraftments onto injured mouse colon epithelium 78
Abstract 79
I. INTRODUCTION 81
II. MATERIALS AND METHODS 84
1. Animals 84
2. Scaffolds 84
3. Preparation of EGFP colonoids from mouse colons 85
4. EDTA colonic epithelial injury mouse modeling 86
5. EGFP colonoid transplantation into recipient mouse colon with scaffolds 86
6. Physical assessment after transplantation of colon 87
7. Secondary EGFP colonoid formation analysis 88
8. Cytotoxicity assay of colonoids with scaffolds 88
9. Antibodies used for immunohistochemistry 89
10. Reverse transcriptase-polymerase chain reaction analysis 89
11. Immunofluorescence 91
12. Statistical analysis 91
III. RESULTS 92
1. Colonoids characterization 92
2. In vitro toxicity testing of scaffolds for colonoids 94
3. Evaluation of colonoid engraftment efficiency by scaffolds 98
4. Scaffold mediated enterotoxigenicity in colonoid transplantation 109
5. Analysis of long term colonoid engraftment by scaffold 112
IV. DISCUSSION 117
V. REFERENCES 120
ABSTRACT IN KOREAN 128
PART III. Development of Collagen Based 3D Matrix for Gastrointestinal Tract-Derived Organoid Culture 130
Abstract 131
I. INTRODUCTION 133
II. MATERIALS AND METHODS 135
1. Materials 135
2. Animals 135
3. Preparation of collagen-based matrix for organoid culture 136
4. Isolation and culture of mouse colon organoids 136
5. Isolation and culture of mouse small intestinal organoid 137
6. Isolation and culture of mouse stomach organoid 137
7. Isolation and culture of human colon organoid 138
8. EGFP mouse derived colon organoid transplantation into recipient mouse colon 139
9. Quantitative reverse transcriptase-polymerase chain reaction analysis 140
10. WST-1 activity assay 141
11. Immunofluorescence 141
12. Statistical analysis 142
III. RESULTS 143
1. Optimization of collagen matrix culture conditions using mouse colon crypt 143
2. In vitro mouse colon organoids culture in collagen matrix 149
3. In vitro mouse small intestinal organoids cultured in collagen matrix 153
4. In vitro mouse stomach organoid cultured in collagen matrix 157
5. In vitro human colon organoid cultured in collagen matrix 160
6. In vivo EGFP+ transplantation of mouse colon organoid cultured in collagen matrix[이미지참조] 163
IV. DISCUSSION 166
V. REFERENCES 169
ABSTRACT IN KOREAN 173
GENERAL CONCLUSION 175
PART I 10
Table 1. Antibody list for immunohistochemistry 42
Table 2. Primer list for RT-qPCR 63
PART II 10
Table 1. Success rate of transplantation at 7 days post transplantation 105
PART I 11
Fig. 1. Modeling of radiation proctitis to evaluate efficacy of colon organoids 29
Fig. 2. Engraftment of colon organoids into the irradiated rectal mucosa 36
Fig. 3-1. Colon epithelium regeneration of radiation induced rectal damage by... 41
Fig. 3-2. RNA-Seq for epithelial cells derived from colon organoid engraftment tissues 44
Fig. 3-3. Secondary organoid formation assay in engraftment tissues 49
Fig. 3-4. Endoscopy analysis of the radiation proctitis regeneration after colon... 52
Fig. 3-5. Functional regeneration of radiation induced rectal damage by colon... 55
Fig. 4. Lgr5+ stem cells are a key treatment factor for regeneration effects of...[이미지참조] 58
Fig. 5. Development of endoscopic transplantation and cryopreseraction of colon... 66
PART II 12
Fig. 1. Characterization of EGFP+ colonoids from CAG-EGFP mice[이미지참조] 93
Fig. 2. In vitro cytotoxicity assay in colonoid culture with scaffolds[원문불량;p.80] 95
Fig. 3-1. Schematic depiction of the protocol used to generate EGFP+ colonoid...[이미지참조][원문불량;p.84] 100
Fig. 3-2. Quantitative analysis of EGFP+ engraftment with each scaffold[이미지참조][원문불량;p.87,89] 103
Fig. 3-3. Secondary colonoid formation assay[원문불량;p.92] 108
Fig. 4. Physical analysis of GFP+ colonoid transplanted mouse colon 111
Fig. 5. Long term tissue analysis of EGFP+ engraftment of each scaffolds[이미지참조][원문불량;p.98] 114
PART III 12
Fig. 1. Effect of the components of collagen-based matrix on culturing mouse... 145
Fig. 2. Maintenance of mouse colon organoids in Matrigel and in Collagen-based... 150
Fig. 3. Comparison of growing mouse small intestinal organoids in Matrigel and... 156
Fig. 4. Comparison of growing mouse gland-derived stomach organoids in... 159
Fig. 5. Comparison of growing human-derived colon organoids in Matrigel and in... 162
Fig. 6. Transplantation of collagen matrix-cultured colon organoids into recipient... 165