臺灣博碩士論文加值系統

單細胞分析對於醫療科學和生物研究扮演了相當重要的角色，但是從檢體中取出高純度的稀少細胞是相當嚴峻的挑戰。許多文獻顯示，循環腫瘤細胞數量對於病人癒後有明顯的關係。但這種細胞非常稀少且異質性高，為了更加了解循環腫瘤細胞於癌症轉移的過程中所具有的臨床價值，對於這些細胞進行分析及研究開始受到重視。本論文展示了一套搭配微流道碟盤系統的單細胞抓取系統，藉由改良原有的設計來達到節省細胞抓取的時間的情況下並不會降低細胞的回收率。MCF7乳癌細胞株以及白血球被用來展示此系統在多顆細胞抓取上的高抓取率以及回收率並不受到不同細胞形態的影響，從結果顯示利用MCF7以及白血球做實驗，回收率可以達到平均95%的水準，為了滿足不同的細胞密度的條件，系統中搭配了特殊的設計來提高在細胞密度提高的情況下仍能維持抓取的回收率以及純率。除此之外，利用融合瘤細胞的單株化抗體實驗來證明對於細胞的生長以及功能性也不會造成太大的影響，除了細胞分析外，此系統仍可應用於單細胞的培養上，藉由比較分選後的細胞和原有的細胞株比較抗體的效價來佐證此單細胞抓取系統並不會影響細胞的成長和功能。藉著特殊設計平台的幫助可以實現不同需求的自動化，改善自動化的流程將一次抓取單顆細胞改良成抓取12顆細胞來達到減省了近68%的細胞抓取時間，同時具有高抓取率以及回收率，可望成為一套有利的系統來協助單細胞分析以及細胞培養的後端研究。

Many studies have reported poor disease progression due to high number of detected circulating tumor cell (CTC). To advance the understanding and clinical value of CTC in metastasis progression, profiling and analyzing of CTC is highly desirable. Hence, single cell analysis plays an important role in medical research and biological applications. However, retrieval of rare cells from blood with high purity is highly challenging.
This thesis presents a single cell retrieval system to remove target single cells from heterogeneous population of cells. MCF7 and WBC were used to demonstrate the system’s accuracy on different cell types and the effect of cell density on retrieval rate. Moreover, the microcavity chip were used to enhance retrieval rate. B-cells fused with melanoma which called hybridoma were used to demonstrate the effect of retrieval on cell function. CTC was retrieved from whole blood. After enrichment CTC with microfluidic disk system developed in our laboratory, single cell retrieval system can retrieve CTC from collecting chip. Results show the cell retrieval rate reach an average of 95% for cell lines and time saving of 68% than original procedure at 103 cells/ml without microcavity chip. When the cell density in the microcavity chip increases to 104 cells/ml, the retrieval rate is still at an average of 95%. Moreover, retrieval of high purity CTC from whole blood was successful. The results of ELISA of hybridoma confirm that there is no significant effect of single cell retrieval system on cell function. In conclusion, the single cell retrieval system has realized the primary automation of cell retrieving technique with high retrieval rate and retrieval purity, which should become a powerful tool to assist single cell analysis and culture of single cell.

目錄Table of Contents
口試委員會審定書 1
致謝 2
中文摘要 3
圖目錄 List of figures 7
表目錄 List of tables 8
Chapter 1. Introduction 9
1.1 The importance of single cell analysis in medical & biological applications 9
1.2 Existing technologies of single cell retrieval 10
1.3 Previously developed of single cell retrieval system in our laboratory 13
1.4 Current single cell retrieval system 15
Chapter 2. Architecture of single cell retrieval system 16
2.1 System architecture overview 16
2.2 Design concept 17
Chapter 3. Material and methods 19
3.1 Fabrication of the micropipette tip 19
3.2 Cell culture & medium preparation 20
3.3 Whole blood sample preparation 21
3.4 Experimental set-up 22
3.5 Cell detection 23
3.6 Procedures for automated single cell retrieval 25
Chapter 4. Results and discussion 28
4.1 Characterization of the single cell retrieval system 28
4.2 Retrieval efficiency and accuracy on different cell types 28
4.3 Parameters optimization 32
4.4 Compare between new procedure and original procedure 37
4.5 Application of system’s versatility on medical & pharmaceutical 38
Chapter 5. Concluding Remarks 43
References 44

[1]Hayes, D.F., et al., Circulating tumor cells at each follow-up time point during therapy of metastatic breast cancer patients predict progression-free and overall survival. Clinical Cancer Research, 2006. 12(14): p. 4218-4224.
[2]Cristofanilli, M., et al., Circulating tumor cells, disease progression, and survival in metastatic breast cancer. New England Journal of Medicine, 2004. 351(8): p. 781-791.
[3]Cohen, S.J., et al., Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer. Journal of Clinical Oncology, 2008. 26(19): p. 3213-3221.
[4]Bidard, F.C., et al., Clinical validity of circulating tumor cells in patients with metastatic breast cancer: a pooled analysis of individual patient data. Lancet Oncology, 2014. 15(4): p. 406-414.
[5]Alix-Panabieres, C. and K. Pantel, Technologies for detection of circulating tumor cells: facts and vision. Lab on a Chip, 2014. 14(1): p. 57-62.
[6]Harouaka, R., et al., Circulating tumor cells: Advances in isolation and analysis, and challenges for clinical applications. Pharmacology & Therapeutics, 2014. 141(2): p. 209-221.
[7]Groebe, A., et al., Prognostic Relevance of Circulating Tumor Cells in Blood and Disseminated Tumor Cells in Bone Marrow of Patients with Squamous Cell Carcinoma of the Oral Cavity. Clinical Cancer Research, 2014. 20(2): p. 425-433.
[8]Frumkin, D., et al., Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues. Bmc Biotechnology, 2008. 8: p. 16.

[9]Cristofanilli, M., et al., Circulating tumor cells, disease progression, and survival in metastatic breast cancer. New England Journal of Medicine, 2004. 351(8): p. 781-791.
[10]Huang, R., et al., A microfluidics approach for the isolation of nucleated red blood cells (NRBCs) from the peripheral blood of pregnant women. Prenat Diagn, 2008. 28(10): p. 892-9.
[11]Fuchs, A.B., et al., Electronic sorting and recovery of single live cells from microlitre sized samples. Lab on a Chip, 2006. 6(1): p. 121-126.
[12]Peeters, D.J.E., et al., Semiautomated isolation and molecular characterisation of single or highly purified tumor cells from CellSearch enriched blood samples using dielectrophoretic cell sorting. British Journal of Cancer, 2013. 108(6): p. 1358-1367.
[13]Ding, X., et al., Standing surface acoustic wave (SSAW) based multichannel cell sorting. Lab Chip, 2012. 12(21): p. 4228-31.
[14]Nam, J., et al., Separation of platelets from whole blood using standing surface acoustic waves in a microchannel. Lab Chip, 2011. 11(19): p. 3361-4.
[15]Frumkin, D., et al., Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues. BMC Biotechnol, 2008. 8: p. 17.
[16]Kasukurti, A., et al., Single-cell isolation using a DVD optical pickup. Optics Express, 2011. 19(11): p. 10377-10386.
[17]Lu, Z., et al., Single Cell Deposition and Patterning with a Robotic System. Plos One, 2010. 5(10): p. 9.
[18]Kornyei, Z., et al., Cell sorting in a Petri dish controlled by computer vision (vol 3, 1088, 2013). Scientific Reports, 2013. 3: p. 1.

[19]Anis YH, Holl MP and Meldrum DR (2010) Automated selection and placement of single cell using vision-based feedback control. IEEE Trans Automat Sci Eng 7: 598–606.
[20]Peeters, D.J.E., et al., Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting. British Journal of Cancer, 2013. 108(6): p. 1358-1367.