細胞培養技術在當今的基礎與臨床醫學研究領域上扮演了不可或缺的角色。利用細胞培養皿進行細胞培養實驗是當今最主流且被廣泛使用的細胞培養方式，其具有容易操作及觀察等特性，然而此種技術係將細胞培養於二維平面上，與生物體內三維細胞成長環境相比尚具有極大的不同。因此近年來，三維細胞培養越來越受到重視。有許多研究證實三維培養的細胞在型態、蛋白質及基因表現或抗藥性等特性上與二維培養細胞相比具有很大的不同。因其培養環境更接近於生物體內的狀況，在三維培養環境中進行的藥物測試，相較於二維細胞培養實驗能提供更好的預測結果。在不同的三維細胞培養技術中，懸掛液珠法因具有了容易操作、成本低廉等特性而被廣泛使用。然而要利用懸掛液珠進行細胞藥物測試，必須以人工逐一處理每顆懸掛液珠，或以機械手臂進行高通量測試，因此具有很高的時間及金錢成本。微流體科技因能夠精準的操控小量液體，並且在單位面積上提供大量的測試，在細胞培養與藥物測試方面可以提供良好的解決方案。然而目前利用微流體技術應用於懸掛液珠細胞培養之藥物測試，須藉由外部儀器或閥門結構來產生藥物測試所需的濃度序列，操作上相對困難。因此在本論文中，提出一種用於整合懸掛液珠三維細胞培養與藥物測試的簡易解決方案。此種方法僅須使用實驗室常見之微量移液器，即可操作晶片產生三維細胞培養環境，並且藉由簡易滑動產生藥物濃度序列應用於藥物測試。此外，不同於之前之滑動微流體晶片，藉由製造晶片所用的原料改良，可讓此晶片之可操作時間延長，在至少一周之長時間細胞培養後仍具有可操作之的能力，更能夠符合不同細胞藥物測試時間之需求。

Cell culture systems are indispensable tools that are used in a wide range of laboratory and clinical researches. Using petri dish to perform cell culture experiments has been a well-developed and common method for cell biology research. However, in last decades, three-dimensional (3D) cell culture systems have gained more and more interest due to their advantages in providing more physiologically relevant information and more predictive data for in vivo tests. In a variety of 3D cell culture methods, hanging drop system has been widely used for 3D cell sphere culture application. This method traditionally requires manual or robotic arm to operate, thus it is time consuming or high cost. Microfluidic technology provides an attractive tool due to its ability of handling low volume liquid and potential of building high-throughput systems. Since 3D cell culture provides a closer microenvironment to human body, performing drug discovering based on it bear the potential of more predictive power for clinical efficacy than on 2D. However, till now, the drug testing relevance of 3D cell culture systems mainly require control valves or pumps to drive the fluid, which increases the complexity of operation and limits the application. In this project, we present a new approach which can be used to build a simplified hanging drop chip system integrated with drug testing function. Without using other external equipment, three-dimensional cell culture and drug application processes can be easily done by using a conventional pipette and sliding the chip. The material for making the chip is also modified from traditional microfluidic materials, which allows the ability of further operation after culturing cell up to 7 days.

摘要-----------------------------------------------------------------------------I
Abstract------------------------------------------------------------------------II
Table of contents--------------------------------------------------------------III
Literature Review----------------------------------------------------------------1
Two-dimensional (2D) vs. three-dimensional (3D) cell cultur----------------------1
Hanging drop method on microfluidic device---------------------------------------4
Concentration gradient generator on microfluidic device--------------------------8
Slip chip technique-------------------------------------------------------------10
Materials and methods-----------------------------------------------------------11
Chip design---------------------------------------------------------------------11
Chip fabrication----------------------------------------------------------------16
Device substrate and holder-----------------------------------------------------21
Numerical simulation------------------------------------------------------------22
Comparison of the long-term lubrication ability of normal and modified PDMS-----22
Cell culture and maintenance----------------------------------------------------24
Cell toxicity test--------------------------------------------------------------24
Cell imaging--------------------------------------------------------------------27
Experimental Results------------------------------------------------------------28
Device fabrication--------------------------------------------------------------28
Device assembly-----------------------------------------------------------------31
Characterization of modified PDMS for device fabrication------------------------33
Chip operation------------------------------------------------------------------35
Drop formation height measurement-----------------------------------------------36
Aggregation test----------------------------------------------------------------37
Time-dependent concentration by diffusion---------------------------------------38
Cell culture experiment---------------------------------------------------------41
Discussion----------------------------------------------------------------------46
Conclusion----------------------------------------------------------------------47
References----------------------------------------------------------------------48

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