早期偵測循環性癌細胞與它的細胞表面標誌可提供癌症診斷標靶治療重要的訊息。直到現在，抓取並偵測循環性癌細胞的方法既複雜又昂貴，而且抓取到的細胞無法直接培養放大數量。循環性癌細胞很稀少，在十億的血球細胞中才出現幾顆，因此需要一個方便又便宜的方法有效地抓取並培養循環性癌細胞。
此篇論文利用微載體Cytodex 1結合細胞過濾設備抓取並培養循環性癌細胞。過濾膜會先塗上一層polyHEMA，防止循環性癌細胞貼附，以得到較好的回收率。癌細胞貼附在微載體後，血球細胞被洗掉，加入培養液到細胞過濾設備以培養增殖循環型癌細胞。增殖後的癌細胞將可進行多重分析，像是免疫染色、冷光分析和聚合酶連鎖反應。為了容易觀察微載體抓到的循環性癌細胞，此研究也設計和製造微流道晶片，並利用PEGDA水膠在晶片裡建立微結構，以逐一地捕獲並排列微載體。藉由在過濾裝置裡簡單地混合Cytodex 1和HCT-8細胞株，研究顯示Cytodex 1可以有效地抓取並培養癌細胞。在細胞與微載體混合四小時之後，細胞抓取效率可達86%。此技術也可用於偵測癌細胞表面標誌如表皮細胞貼附分子、細胞角蛋白19和定出細胞數量。而微流道晶片中的微結構也成功地將微載體排列以利後續觀察或其他可能分析之使用。總結而言，本研究提供簡易的循環性癌細胞抓取、培養和分析，既經濟且方便，未來可望發展成癌症臨床診斷和預後的工具。

Early detection of circulating tumor cells (CTCs) and their cell surface marker analysis provide critical information for cancer diagnosis and target therapy. Until now, CTC capture and detection methods are complicated, costly and the captured cells could not be cultured and expanded directly. Because CTCs are rare, existing at only a few per one billion blood cells, a highly efficient method is required to capture and culture CTCs for further assay. This thesis used microcarrier beads Cytodex 1 combining a Cell Strainer device to capture and culture CTCs. The filter membrane in the Cell Strainer was first coated with poly 2-hydroxyethyl methacrylate to prevent CTC from binding to achieve better isolation efficiency. After cancer cells have bound to microcarrier beads and blood cells were removed, culture medium was added to the Cell Strainer device to expand the CTC. The feasibility of using the expanded cancer cells in multiple analyses, such as immunostaining, luciferase assay, and PCR were determined. Cyotdex 1 has been shown to effectively capture and culture HCT-8 cells by simply mixing them in the Cell Strainer device in a well. Cell capture efficiency with Cytodex beads can reach 86% after 4 h of cell seeding. This technique was followed by immunostaining the cancer cell marker epithelial cell adhesion molecule EpCAM, RT-PCR analysis the gene marker cytokeratin-19, as well as to quantify the cell number using an ATP-based luminescence assay. To simplify the observation of the captured CTC on the microcarrier beads, this study also designed and fabricated a microfluidic chip using polyethylene glycol diacrylate hydrogel to create microstructures. The chip could trap and arrange the microcarrier beads individually. The microstructure successfully arranged microcarrier beads in an array for easy observation and other potential uses. In summary, the method demonstrated in this thesis provides a useful tool for simple CTC capture, culture and analysis and have the advantages of economical and convenient, and may be applied in clinical diagnosis and prognosis in the near future.

中文摘要 Ⅰ
Abstract Ⅱ
Acknowledgement Ⅲ
Table of Contents Ⅴ
List of Figures Ⅷ
Abbreviations Ⅸ

Chapter 1 Introduction 1
Chapter 2 Materials and Methods 6
2.1 Cell culture 6
2.2 Microcarrier cell culture. 6
2.3 Preparation of polyHEMA-coated plate and membrane 6
2.4 Antibody conjugation on microcarrier beads 7
2.5 Capture efficiency determination 7
2.6 Cell viability determination 8
2.7 Immunofluorescence staining 9
2.8 Reverse transcription-PCR 9
2.9 Determination of cell number by ATP-regeneration luciferase assay 10
2.10 Circulating tumor cell captured from the blood samples of tumor bearing mice 11
2.11 Cell density determination by alamar blue assay 11
2.12 Spheroid formation 12
2.13 Fabrication of a PDMS microfluidic chip for microcarrier beads separation 12
2.14 Glass surface treatment with TMSPMA 13
2.15 Fabrication of PEG-DA hydrogel-based microstructure 13
2.16 Microcarrier beads trapping by the microstructures 14
Chapter 3 Results 15
3.1 Effects of polyHEMA coating on cell strainer membrane on cell
attachment 15
3.2 Microcarrier cell culture with microcarrier beads using polyHEMA coated plates 15
3.3 Cell capturing on antibody-conjugated microcarrier beads 15
3.4 Cell captured efficiency and cell viability with microcarrier beads 16
3.5 Immunofluorescence staining of cells on microcarrier beads 16
3.6 Detection of cancer marker CK19 with RT-PCR 17
3.7 Enumeration of the cell number using ATP-regeneration based luminescence assay 17
3.8 Capture circulating cancer cells from the blood of mice carrying tumor xenografts 18
3.9 Comparison of cell density in a fixed area 18
3.10 PEG microposts arranged microcarrier beads in the microfluidic chip 19
Chapter 4 Discussion 20
References 24
Tables 32
Figures 33

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