癌症為連續第33年高居台灣十大死因首位，平均每100人就有28人死於癌症，癌症在治療上的高困難度是因為癌細胞的轉移所導致。在轉移的過程中會產生「循環腫瘤細胞」（Circulating Tumor Cell, CTC）。循環腫瘤細胞是指從原生腫瘤或轉移性腫瘤脫離並進入血液循環系統的腫瘤細胞。研究中顯示循環腫瘤細胞的多寡與病人的存活率和病情的預測有正相關，因此偵測和計數循環腫瘤細胞是對於癌症治療相當重要的，特別是對早期癌症轉移和接受藥物治療長期追蹤的病人。
本論文中所使用的觀測晶片，是在載玻片上以SU-8做出結構，利用流體的特性，使懸浮細胞液內的細胞受液體流動的拉力而產生單層緊密排列的自組裝排列。另外，本論文中所使用的染色晶片，是使用高分子材料 Polydimethylsiloxane (PDMS) 上做出薄膜以及流道結構，PDMS 薄膜上含有孔洞陣列，用來乘載細胞液與螢光染劑。實驗室與台北榮民總醫院合作取得大腸癌第四期病患的血液檢體，未來設計的實驗流程為經過血液分離前處理後，將檢體放入染色晶片中，利用流體擴散的方式，使薄膜上層細胞液內多餘的螢光染劑因濃度差而擴散至底層流道，完成染色標定。將檢體放入觀測晶片中，利用重力和側向拉力的影響，在觀測區域形成二維陣列緊密排列。以螢光顯微鏡觀測循環腫瘤細胞並計算其數量再和流式細胞儀的結果對比。
除了能自組裝產生單層緊密的排列，也能在晶片上完成染色的程序，使檢測能更加快速、便利。

High degree of difficulty in the treatment of cancer is due to cancer metastasis. In the transfer process, it will produce "circulating tumor cells" (Circulating Tumor Cell, CTC). Studies show the amount of circulating tumor cells is correlated to probability of survival of patient.
As the number of CTCs is quite few, we expect to undergo a gentle way in detection process in order not to lose too many CTCs during the sample preparation process. In the traditional way of staining process by mixing and centrifugation procedures, not only a long process (1.5 hours for 3 dyes) is usually required but also the centrifugation force is harmful for cell viability. To solve this issue, a Three-Dimensional Microwell Perfusion-Array is designed in this study to perform gentle fluorescence-removal process by diffusion-based flow processes on CTCs and WBCs without centrifuging. In this study, clinical whole blood samples from three VI TNM stage cancer patients were tested on SACA chips [1] after pre-centrifuging out erythrocytes and staining.

第1章 緒論 1
1 .1 研究背景 1
1 . 2 研究目標 5
第二章 文獻回顧 6
2 .1 細胞篩選方法 6
2 .1 .1 CellSearch系統檢測循環腫瘤細胞 6
療效評估 8
癒後判斷 8
2 .1 .2 免疫標定(Immunolabeling) 9
流式細胞儀(Flow Cytometer) 9
2 .1. 3非免疫標定(Non-immunolabeling) 15
第三章 實驗設計 18
3 .1 螢光檢測系統特色 18
3 . 2 實驗材料準備 19
3 .2 .1 細胞培養 19
3 .2 .2 藥品介紹 20
3 .2 .3 藥品製備處理 23
3 .3 微流道井式排列平台 25
3 .3 .1 微流道井式排列平台設計及原理 26
3 .3 .2 平台製成及組裝 28
3. 4. 轉移大腸癌病患檢體於井式細胞排列平台檢測 31
3. 4. 1病患檢體於自組裝細胞排列平台檢測結果 31
3 .5螢光染色三維擴散晶片 34
3. 5. 1螢光染色三維擴散晶片設計和原理 35
3. 5. 2螢光染色三維擴散晶片製程和組裝 38
第四章 實驗結果與討論 42
4. 1 個別螢光染劑染色 42
4. 1. 1 Hoechst 標定 BT474 cell line 42
4. 1. 2 CD45 - PECy7 標定白血球 45
4. 1. 3. EpCAM – FITC 標定 BT474 cell line 48
4.2 全血加入cell line 螢光染劑染色 54
第五章 總結 58
5. 1 未來工作 60
第六章 參考文獻 61

第六章 參考文獻
1. Tsung-Ju Chen, High-efficiency rare cell identification on a high-density self-assembled cell arrangement chip. Biomicrofluidics, 2014, 036501
2. Klein, C.A., Parallel progression of primary tumours and metastases. Nature Reviews Cancer, 2009. 9(4): p. 302-312.
3. Christine L., A Perspective on Cancer Cell Metastasis. Science review, 2011. 3.
4. Timothy A., et al., Circulating Tumor Cells: A Multifunctional Biomarke., Clinical Cancer Research, 2014.5.
5. Klaus Pantel1 and Catherine Alix-Panabieres, Circulating tumour cells in cancer patients: challenges and perspectives. Trends in Molecular Medicine Vol.16 No.9, 2010.
6. John D. O’Flaherty, et al., Circulating tumour cells, their role in metastasis and their clinical utility in lung cancer. Elsevier, 2012.
7. Krebs, M.G., et al., Evaluation and prognostic significance of circulating tumor cells in patients with non–small-cell lung cancer. Journal of Clinical Oncology, 2011. 29(12): p. 1556-1563.
8. Miller, M.C., G.V. Doyle, and L.W. Terstappen, Significance of circulating tumor cells detected by the CellSearch system in patients with metastatic breast colorectal and prostate cancer. Journal of oncology, 2009. 2010.
9. Hoffman, R. and W. Britt, Flow-system measurement of cell impedance properties. Journal of Histochemistry & Cytochemistry, 1979. 27(1): p. 234-240.
10. Weian Sheng, et al, Capture, release and culture of circulating tumor cells from pancreatic cancer patients using an enhanced mixing chip. Lab Chip, 2014, 14, 89-98
11. Shutao Wang. et al, Highly Efficient Capture of Circulating Tumor Cells by Using Nanostructured Silicon Substrates with Integrated Chaotic Micromixers. Angew. Chem. Int. Ed. 2011, 50, 3084 –3088
12. Shannon L. Stott, et al., Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. PNAS,2010.10. vol 107 p. 18392-18397.
13. Gossett, D.R., et al., Label-free cell separation and sorting in microfluidic systems. Analytical and bioanalytical chemistry, 2010. 397(8): p. 3249-3267.
14. Ting Huang, et al, Highly sensitive enumeration of circulating tumor cells in lung cancer patients using a size-based filtration microfluidic chip. Biosemsors and Bioelectronics, 51, 2014, 213-218
15. Xiaoyun Fan, et al, A microfluidic chip integrated with a high-density PDMS-based microfiltration membrane for rapid isolation and detection of circulating tumor cells. Biosemsors and Bioelectronics, 71, 2015, 380-386
16. Masahito Hosokawa, et al., Size-Based Isolation of Circulating Tumor Cells in Lung Cancer Patients Using a Microcavity Array System. PLoS One. 2013 Jun ;8(6):e67466.
17. Han Wei Hou, et al., Isolation and retrieval of circulating tumor cells using centrifugal forces. SCIENTIFIC REPORTS, 2013.2.
18. Min Yu, et al., Circulating tumor cells: approaches to isolation and characterization. JCB, 2011, volume 192, number 3.
19. Marianna Alunni-Fabbroni., et al., Circulating tumour cells in clinical practice: Methods of detection and possible characterization. Elsevier, 2010.
20. Chang, J.-C., et al. In-parallel rare cells identification by high throughput cells self-assembly. in Nano/Micro Engineered and Molecular Systems (NEMS), 2013 8th IEEE International Conference on. 2013. IEEE.
21. Igor Cima., et al., Label-free isolation of circulating tumor cells in microfluidic devices: Current research and perspectives. Biomicrofluidics, 2013, 7, 011810.
22. Sunitha Nagrath, et al., Isolation of rare circulating tumour cells in cancer patients by microchip technology. NIH Public Access, 2007, 450(7173): 1235–1239.
23. L. G. Rigat-Brugarolas., et al., Highly hydrophilic microfluidic device prototyping using a novel poly(dimethylsiloxane)-based polymeric mix. The Royal Society of Chemistry, 2015, 5, 7423–7425