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研究生:馬維遠
研究生(外文):Wei-Yuan Ma
論文名稱:微流體技術應用於個人化醫療-從腫瘤細胞的體外藥物測試到循環癌細胞的偵測與抓取
論文名稱(外文):Microfluidic Toolbox for Personalized Medicine – from Ex-vivo Drug Testing of Solid Tumor to Detection and Retrieval of Circulating Tumor Cell
指導教授:胡文聰胡文聰引用關係
口試委員:許友恭李黛苹江宏仁許聿翔
口試日期:2015-05-06
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:應用力學研究所
學門:工程學門
學類:機械工程學類
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:83
中文關鍵詞:微流體循環腫瘤細胞單細胞抓取化療敏感性檢測個人化癌症治療
外文關鍵詞:MicrofluidicsCirculating tumor cellsSingle cell retrievingChemosensitivity profilingPersoalized medicine
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循環腫瘤細胞於現階段被認為是一種具有檢測與診斷癌症潛力的指標。許多研究已驗證了循環腫瘤細胞的數量與患者的存活率有明顯的關係,但這種細胞本身極度稀少且異質性高,而現有大部份針對其純化之技術的靈敏度還需要被佐證的狀況下,這些循環腫瘤細胞計數的資訊目前於臨床上並不具有實質的效用。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,對這些細胞進行定性分類、單細胞分析與分子分析的研究開始受到關注。本文展示一套搭配特殊設計平台的細胞回收與抓取系統,細胞株實驗結果證實,以密度將細胞分離的回收技術基礎之下,其細胞回收率不受細胞EpCAM表現量影響,平均回收率為89.7±4.7%,系統靈敏度以混入不同數量級細胞株(10-300顆)得到驗證。細胞抓取系統在適當的參數設定下其對於目標細胞的成功抓取率幾近100%且其抓取純率超越90%。
另一方面,為了因應對於稀少細胞如循環癌細胞或是檢體量受限情況之下的後續研究,本文設計並開發出利用微流體技術的孔盤,能將細胞在繁複的生物相關操作下有效保存高達99.2%,所需之檢體細胞量亦相較於傳統方式減少許多且無損測試的正確性。本文將其應用在癌細胞的藥物敏感度檢測上,經由細胞株(MCF7 and MDA-MB-231)以及實際臨床腫瘤檢體實驗後,驗證了此微流孔盤在細胞數量極少的情況下進行生物性測試實驗的可行性。藉由本研究所發展的全血中循環癌細胞回收系統以及微流孔盤,期望可以應用於癌症的診斷與治療,以提供臨床醫師在無論是病人的癌症管理方面或是藥物選用方面上有效的判斷依據。


Circulating tumor cell (CTC) now is considered as promising biomarker for cancer diagnosis and prognosis. Many studies had reported the overall survival of patients was related to the number of CTCs detected in whole blood. However, patient-derived CTC count may not enough for doctors to make an actionable decision since CTC possess high heterogeneity, yet the sensitivity of current technologies for CTC enrichment remains to be validated. To further investigate the role of CTC in metastasis process, there has been great interest in subtyping, single cell profiling and molecular analysis of CTC recently. This thesis presents a microfluidic-based, automatic, seamless system enabling single CTC isolation & retrieval from whole blood sample. Cell line results of isolation system showed the ability to recovery CTCs with 89.7 ± 4.7% efficiency from 2ml of human whole blood regardless of their EpCAM expression level. Sensitivity is achieved at low concentrations and recovery of cells through the range of 10 to 300 cells. Results of single cell retrieval system showed the successful retrieval rate reached nearly 100% of target cells and the average retrieval purity was over 90%.
On the other hand, for downstream cellular assay on rare cell or limited cell-supplied sample, this thesis presents a microfluidic-based, 96well-formatted plate with built-in micro gap to preserve these precious cells up to 99.2% during multiple assay/wash procedures. Comparing to traditional plate much less cell was needed when using this plate. Drug response profiling on limit cell-supplied tumor sample was performed as one application of this plate. Results on cell line (MCF7 and MDA-MB-231) and patient tumor sample showed the feasibility of cellular analysis in rare cell scenario using this plate.
In this thesis, an automatic CTC isolation & retrieval system and a micro-gap plate for cellular analysis of rare cell were presented. Solid evidence of feasibility, stability, and robustness of this whole system were also provided. Conceivably, this microfluidic-based system is applicable to personalized medicine, providing patient-derived information for doctors to tailor therapeutic interventions for patients.


謝辭………………………………………………………………………………………i
中文摘要………………………………………………………………………………..ii
Abstract…………………………………………………………………………………iii
Contents………………………………………………………………………………..v
圖目錄…………………………………………………………………………………vii
表目錄…………………………………………………………………………………viii
1. Introduction …………………………………………………………………………...1
2. System overview………………………….…………………………………………12
2.1. Schematic workflow of single cell isolation and retrieval from whole blood..12
2.2. Automated system for CTC isolation, collection and IF staining…………….13
2.3. Image-based automatic target detection software…………………………….16
2.4. Automated single cell retrieval system……………………………………….18
2.5. Design concept of Micro-Gap Plate (MGP) for rare cell and low cell-supplied tumor sample study……………………………………………………………20
3. Materials and Methods………………………………………………………………23
3.1. Fabrication of disk and microcavity chip…………………………………….23
3.2. Fabrication of micropipette tip……………………………………………….24
3.3. Cell lines and cell culture…………………………………………………….25
3.4. Procedures for cell retrieval…………………………………………………..25
3.5. Fabrication and sterilization of MGP………………………………………...26
3.6. Characterization of MGP…………………………..……………………........28
3.7. Preparation of tumor samples………………………………………………...32
3.8. Flow cytometry analysis of tumor samples………………………………….33
3.9. Immunohistochemistry of tumor samples (ER, PR and Her2/neu)…………33
3.10. Procedures of drug response profiling using MGP……………………34
3.11. Procedures of drug response profiling in control (96-well plate)……36
3.12. Statistical analysis…………………………………………………………37
4. Results and discussion……………………………………………………………….38
4.1. Performance of CTC isolation system………………………………………..38
4.2. Characterization of microcavity in the cell collecting chip…………………..39
4.3. Trapping efficiency of microcavity…………………………………………..42
4.4. Automatic cell retrieval in microcavity chip with whole blood processing….46
4.5. Viability studies of retrieved cells with whole blood processing……………49
4.6. Cell conservation after solution exchange……………………………………50
4.7. Stability of solution concentration during long-term incubation…………….51
4.8. Drug response profiles of cancer cell lines on MGP and 96-well…………..53
4.9. Morphology of MCF7 and MDA-MB-231 cells during treatment…………55
4.10. Minimum requirement of cell number for cell-based assays using the MGP58
4.11. Clinical pathological features of patient samples evaluated………………60
4.12. Morphology of primary cells of patient samples…………………………61
4.13. Drug response profiles of patient samples on MGP………………………65
5. Conclusion…………………………………………………………………………69
6. Acknowledgement…………………………………………………………………..71
7. Supplementary information………………………………………………………72
7.1. The correlation between manual and auto quantification…………………….72
7.2. The viability assessment by MTT assay……………………………………...74
7.3. The tri-staining method………………………………………………………76
8. References…………………………………………………………………………...77


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