本研究主要著重於結合生物及工程領域，期盼利用微流體系統創造非化學作用之裝置，使細胞得於人體外長時間存活。目前微流體系統於生物微奈米科技所遇到之困難為無法結合單一細胞培養，以及取出微量培養液進行後端濃度及分泌物之分析；然而本研究突破此困難，利用不同厚度之聚二甲基矽氧烷(PDMS)薄膜及兩層微井、流道之組成，將細胞固定於特定位置，進行長時間個別培養，並定時取出微量培養液(液珠形成)再行置換，分析細胞之分泌物。此外，非貼附型之人類單核白血球細胞(HL-CZ)以及貼附型之乳癌細胞(MDA-MB-231)為此研究之標靶物，期盼藉由新穎之裝置得以了解兩株細胞之個別表現。研究結果發現培養液是否每天進行不同長度時間之置換對細胞有重大之影響，每天長時間置換培養液將減低細胞之存活率，然而特定時間之置換將拉長細胞之生存天數。有趣的是，由於乳癌細胞對於不同厚度之材料有著敏感之特性，因此研究測試兩種不同厚度之材料，觀察其生長之狀況。研究發現乳癌細胞於較厚之材料較易生存，然而較薄之材料將降低其存活率。此兩株細胞之研究不僅僅可以幫助病患減低測試體內癌症治療之痛苦，也得以幫助了解細胞分泌物對於人體之影響。因此研究對於社會之貢獻為可以提前治療癌症疾病以及傳染病，控制其生存情況並了解分泌物對於人體之嚴重情況，解決多年來於微奈米生物科技所遇到之困難。

Screening cells is often the first step in multitude of in vitro assays modernly used in drug testing and in molecular and cell biology. However, the screening algorithm is often based on detecting the fluorescence intensity, limiting the type of assays conducted on cells. Here, we have developed a microfluidic platform integrating droplet-generation and cell-culture systems for screening cells based on long-lasting and transient responses.
This fully automatic microfluidic device is capable of 1) trapping small cell populations in an array of predetermined locations, 2) inspecting cells in a microscope for long periods of time, and 3) selectively retrieving samples of supernatant fluids in droplets from different cell colonies in individual traps for measurements of metabolites and autocrine/paracrine signaling molecules. Local altering microtopography of transparent elastomeric substrate at addressable locations were used to trap and release small fluid volumes (droplets) and cells.
Immiscible solution was introduced to main channel isolating each cell culture microwell. Droplets of various sizes could be generated by precise control of flow rates in immiscible solution and deflection of elastomeric substrate.
Non-adherent HL-CZ and adherent MDA-MB-231 cell lines were cultured in the device. Results suggested that both cell types could be cultured in isolated microwells for seven days without exchanging cell culture medium; however, exchanging cell culture medium every day for longer times reduced the survival rate.
What’s more, MDA-MB-231 cells appeared sensitive to thickness of the elastomeric substrates. Survival rate of MDA-MB-231 cells was much higher when cultured on elastomeric substrates with 60 micrometer thickness in contrast to 30 micrometer.
In summary, we have developed a microfluidic platform capable of long-term cell culturing and sampling cell culture medium over time, and have a great promise to yield new insights into cell behavior and development.

Abstract...........................................i
Content..........................................iii
List of Figures....................................v
List of Tables..................................viii
Chapter 1 Introduction.............................1
1-1 Preface.....................................1
1-2 Background and Value........................3
1-3 Motivation and Future Prospect..............4
Chapter 2 Literature Review........................5
2-1 T-Channel...................................5
2-2 Controllable Moving-Wall Structure..........7
2-3 Cross-Channel...............................8
2-4 Hanging Drop................................9
2-5 Acoustic Waves.............................12
2-6 Summary....................................14
Chapter 3 Materials and Methods...................15
3-1 Design Concept.............................15
3-2 Soft Lithography...........................16
3-2-1 Pretreatment...........................17
3-2-2 Spin Coat..............................17
3-2-3 Soft Bake..............................17
3-2-4 Exposing...............................17
3-2-5 Post Expose Bake (PEB).................18
3-2-6 Develop................................18
3-2-7 Rinse..................................18
3-2-8 Hard Bake..............................18
3-3 PDMS Bonding...............................19
3-4 Clamp Molding..............................20
3-5 Other structures for PDMS bonding..........22
3-6 Experimental Setups........................24
Chapter 4 Results.................................25
4-1 Membrane formation.........................25
4-2 Membrane Deflection........................27
4-3 Droplet Generation.........................31
4-4 Cell culture medium Replenishment..........34
4-5 Cell Viability.............................36
4-5-1 Introduction to HL-CZ and MDA-MB-231 cell line ..................................................36
4-5-2 Cell Viability: HL-CZ cell..................38
4-5-3 Cell Viability: MDA-MB-231 cell.............44
4-6 Further Discussion.........................53
Chapter 5 Conclusion..............................58
References........................................59

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