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研究生:林璟暉
研究生(外文):Ching-Hui Lin
論文名稱:微流體技術於神經幹細胞應用之發展
論文名稱(外文):Development of microfluidic platforms for neural stem cell research
指導教授:許佳賢許佳賢引用關係
口試委員:王國禎莊志立吳嘉哲劉怡劭
口試日期:2015-07-21
學位類別:博士
校院名稱:國立中興大學
系所名稱:組織工程與再生醫學博士學位學程
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:84
中文關鍵詞:微流體神經幹細胞神經球單細胞單株化培養
外文關鍵詞:MicrofluidicsNeural stem/progenitor cellsneurospheresingle-cellmonoclonal culture
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In this study we aim to develop new platforms for neurosphere assay and single cell manipulation for neural stem/progenitor cell (NSPC) research. The microfluidic cell dissociation platform is designed for enzyme-free dissociation of neurospheres into single-cells. Neurosphere assay is a common method for identification of neural stem/progenitor cells, but obtaining single cells from dissociated neurospheres is difficult using non-enzymatic methods. Our microfluidic chip approach that utilizes flow and microstructures to dissociate neurospheres. Results show that this microfluidic-chip-based neurosphere dissociation method can generate high yields of single cells from dissociated neurospheres of two mouse NSPC models (KT98 and DC115) for 90% and 95%, respectively. The microfluidic chip dissociated cells had high viabilities (80–85%) and the ability to re-grow into neurospheres, demonstrating the applicability of this device to neurosphere-assay applications. In addition to the self-renewal capability, the dissociated cells also retained their normal differentiation potentials, as shown by their capabilities to differentiate into three neural lineages (neurons, astroglia, and oligodendrocytes) when cultured in differentiation culture conditions. Furthermore, neurospheres consist of heterogeneous cell populations including multipotent NSPCs, and lineage immature neuronal and glial precursors and has been shown by the spatial distribution of more differentiated (GFAP+ and TuJ1+) cells at the core and surrounded by the stem cells (Nestin+). The microfluidic cell dissociation platform provides the capability for label-free enrichment of NSPCs from dissociated neurospheres. We demonstrate that the microfluidic chip processed cells show significantly higher NSPC properties such as more SOX2 and FGF1 expression, proliferation rates and neuronal differentiation potential compared to unprocessed cells.
Studying the heterogeneity of single neural cells is crucial but technical difficult. Therefore we develop a cell manipulation method for high-efficiency single cells loading in large microwells. We report the development and application of a dual-well microfluidic device with high-yield of single-cell loading (~77%), long-term single-cell clonal culture capability (7 days) and heterogeneity analysis using single-cell colony formation assay. The high single-cell loading yield is achieved by using sets of small microwells termed “capture-wells” and big microwells termed “culture-wells” according to their utilities for single-cell capture and clonal culture respectively. This novel device architecture allows the size of the “culture” microwells to be flexibly adjusted without affecting the single-cell loading efficiency making it useful for cell culture applications.
We envision that our microfluidic platforms are simple and reliable tools for neural stem/progenitor cells manipulation with high-efficiency and high-throughput properties. In addition to the above advantages, the processed cells from our microfluidic platforms are easy to be used with conventional cell analysis and culture methods for further NSPC investigation and applications.


Acknowledgments I
Table of Content II
List of Tables V
List of Figures VI
Abstract VIII
摘要 X
Chapter 1. Introduction 1
1.1 Motivation 1
1.2 Neural stem/progenitor cell and neurosphere assay 4
1.3 Conventional dissociation methods for obtaining single neural stem/progenitor cells from neurospheres 5
1.4 The heterogeneity of neurosphere component cells 6
1.5 Labeled and label-free system for target cell enrichment 6
1.6 The heterogeneity of single stem and cancer cells 7
1.7 Conventional single cell manipulation methods 7
1.8 Microfluidic device for single cell application 8
1.9 Research Objectives and Specific Aims 10
Chapter 2. Microfluidic Platform Design and Fabrication 12
2.1 Master molds and PDMS replicas fabrication 12
2.2 PDMS Microfluidic Cell Dissociation chip assembling 13
2.3 PDMS microfluidic Dual-Well device assembling 14
Chapter 3. A Mcrofluidic Platform for Neurosphere Research 15
3.1 Enzyme-free dissociation of neurospheres 15
3.1.1 Experimental design 17
Neurosphere formation assay 17
Neurosphere dissociation 18
Calculation of single cell dissociation efficiency from neurospheres 20
Measurement of dissociated cell recovery rate and cell viability 20
Re-sphere assay 21
Induction of dissociated cells 21
Cell imaging 22
Statistical analyses 22
3.1.2 Performances of microfluidic cell dissociation chip platform for enzyme-free dissociation of neurospheres 23
Single cell efficiency of the dissociated neurospheres 23
Cell viability of dissociated neurosphere cells 25
Cell recovery rates of μCDC dissociated neurospheres 26
Self-renewal and differentiation of μCDC dissociated NSPCs 28
3.1.3 Discussion of enzyme-free dissociation of neurospheres 32
3.2 Label-free enrichment of neural stem/progenitor cells from dissociated neurospheres 38
3.2.1 Experimental design 40
Immunocytochemistry staining of neurospheres 40
Enrichment of neural stem/progenitor cells from dissociated neurosphere cells 40
Western blotting 42
Neurosphere proliferation rates of DC and UDC subpopulations 42
3.2.2 Performances of microfluidic cell dissociation chip platform for label-free enrichment of NSPCs from dissociated neurospheres 44
Cellular heterogeneity in neurospheres and NSPCs enrichment efficacy 44
Proliferation and differentiation ability of the high or low stemness subpopulations 48
Chapter 4. A Microfluidic Platform for Single-cell Capture and Culture 51
4.1 High-efficiency single-cell capture and clonal culture of NSPCs and cancer cells 51
4.1.1 Experimental design 54
Cell culture and maintenance 54
DW device preparation for single-cell isolation 54
Single-cell capture and culture 54
NSPCs differentiation in DW devices 57
Cancer cell clonal culture for epidermal growth factor (EGF) promoted colony formation assay in DW devices 57
4.1.2 Performances of microfluidic Dual-Well platform for single-cell capture and clonal culture 59
Single NSPC capture efficiency of DW device 59
Single-cell capture efficiency of different cell types 64
Comparison of single-cell sizes from DW captured cells and native population 68
Small molecule testing in colony formation assay 70
Single-cell clonal culture and NSPC differentiation in the microwells of DW device 72
4.1.3 Discussion of high-efficiency single-cell capture and clonal culture of NSPCs and cancer cells 74
Chapter 5. General Conclusion 77
Chapter 6. Future Works 78
Optimization of single-cell capture efficiency of Dual-well device platform 78
Stable monoclonal cell line establishment using Dual-well platform 78
3D suspension culture by Dual-well platform. 78
References 79


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