建立液動式微型化灌流式細胞培養平台以應用於高通量藥物檢測

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研究生：	賴威翰 LAI, Wei-Han
論文名稱：	建立液動式微型化灌流式細胞培養平台以應用於高通量藥物檢測 Development of Hydraulically-Driven Microperfusion Cell Culture Platform for High Throughput Drug Screening
指導教授：	黃振煌 Huang, Jen-Huang
口試委員:	胡尚秀 Hu, Shang-Hsiu 蕭自宏 Hsiao, Tzu-Hung
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2019
畢業學年度：	107
語文別：	英文
論文頁數：	62
中文關鍵詞：	體外培養模型、藥物篩檢、高通量、微加工技術、灌流系統、液體驅動式微型化灌流式細胞培養系統
外文關鍵詞：	in-vitro models, drug screening, high throughput, micro-fabrication technique, perfusion system, hydraulically-driven micro-perfusion cell culture system
相關次數：	點閱：2 下載：0
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目前於癌症研究以及其藥物之開發上，體外的培養模型儼然已成為重要且標準的工具，例如:於Transwell ®細胞培養皿上進行培養以及三維的細胞球體培養。然而，這些體外培養模型於模擬體內微生理環境時受到了不少的限制，例如:PH值、氧氣濃度、養分供應以及代謝產物的排放等。而近年來，以微流體為基礎而建立的細胞培養平台廣泛地被應用於模擬腫瘤微環境，藉由其灌流系統來運送培養基或是其他生物因子並且精準的控制剪應力以及應變。然而每一個單位的細胞培養裝置都需要搭配著一套專屬的灌流系統以用來模擬微環境。因此，欲進行高通量的培養仍然是一大挑戰。
因此於本研究中，我們透過了結合雷射加工以及逐層建構的微加工技術開發一套具有高通量特性的液體驅動式微型化灌流式細胞培養系統以用來長期培養癌症細胞並且加速癌症藥物的篩檢。每一個培養單元包含了三個主要的原件:細胞培養晶片、培養基儲存槽以及灌流系統。而各單元最終會整合成一平台的型式以便於用一組蠕動幫浦來控制多組灌流單元。藉此，我們可以同時製造出多組有著相同流動情況的脈動流以用來模擬相對應的生理環境。最終，我們要將此細胞培養平台用以更有效地開發癌症治療藥物以及毒性測試。

In vitro culture models including Transwell ® and spheroid have become significant and standard tools for cancer research and drug screening. However, these models have shown the limitations to maintain the physiological levels of the environment, such as pH, oxygen, nutrient supply, and metabolite clearance. Microfluidic-based culture platform recently has been widely used to mimic the tumor microenvironment by precisely controlling the shear stress and mechanical strain, and delivering the reagents and other stimuli to cells when using a perfusion system. Nevertheless, each microfluidic cell culture device requires individual perfusion system to recapitulate the independent microenvironment. Therefore, it is still challenging to operate multiple devices at the same time, especially for high throughput applications.
In this research, we presented a hydraulically-driven micro perfusion cell culture system, which is fabricated by a technique based on the combination of laser engraving manufacturing and additive lamination manufacturing, for long-term cancer cell culture with a high-throughput capability to facilitate the drug screening. Each culture unit mainly combined three parts: a cell culture device, a reservoir for medium storage, and a hydraulically-driven perfusion system. Every culture unit can be assembled into a single platform so that the perfusion systems were able to be controlled by using one single peristaltic pump to generate uniform flow distribution. With this platform, the multiple pulsatile flows can be generated to mimic the physiological relevant environment in each cell culture device. Finally, we aim to use this platform to perform a wide range of tests more effectively for drug development.

Abstract    i
Contents    iv
List of illustrations    vi
List of tables    x
Chapter 1. Introduction and Literature Review    1
1-1    Background    1
1-2    Conventional assay models    2
1-2-1 In vivo models    3
1-2-2 In vitro models    4
1-3    Microfluidic-based culture    6
1-3-1 Microfluidic technology    6
1-3-2 Organ-on-a-chip    9
1-4 Previous study of perfusion-based platform    10
1-5 Purpose    12
Chapter 2. Materials and Methods    13
2-1 Materials    13
2-1-1 PET    13
2-1-2 PMMA    14
2-1-3 PDMS membrane    14
2-2 Platform design and fabrication    16
2-2-1 Drafting and fabrication    16
2-2-2 Setup of micro-perfusion-based cell culture platform    18
2-2-3 Peristaltic pump    20
2-2-4 Micro-pump    21
2-2-5 Micro-valve    23
2-2-6 Bioreactor    25
2-2-7 All-in-one micro-perfusion-based culture platform    27
2-3 Flow characterization    30
2-3-1 Flow rate of the peristaltic pump    30
2-3-2 Flow rate of the micro-perfusion system    31
2-3-3 Stability of the micro-perfusion system    32
2-3-4 Flow pattern of micro-perfusion system    32
2-4 Cell culture    33
2-4-1 Sterilization    33
2-4-2 Cell culture in the culture platform    34
2-5 Drug treatment and assays    36
2-5-1 Treatment on A549/GFP cell    36
2-5-2 Cell viability assay    38
Chapter 3. Current results    39
3.1 PDMS membrane fabrication and thickness selection    39
3.2 Flow characterization experiment    42
3-2-1 Flow rate test of peristaltic pump    42
3-2-2 Flow rate test of the micro-perfusion system    43
3-2-3 Flow stability test    45
3-2-4 Flow pattern of micro-perfusion system    47
3.3 Cell culture in the micro-perfusion-based platform    48
3-3-1 2D static culture in a bioreactor    48
3-3-2 2D dynamic culture in micro-perfusion-based platform    50
3.4 All-in-one micro-perfusion platform    51
3-4-1 Construction of all-in-one micro-perfusion platform    51
3-4-2 Flow rate of all-in-one micro-perfusion device    52
3-4-3 Dynamic cell culture with all-in-one micro-perfusion system    54
3-5 Drug treatment on A549/GFP cell    55
3-5-1 Result of treatment in 96-well plate    55
3-5-2 Dynamic treatment    56
Chapter.4 Conclusion and Future Work    58
4-1 Conclusions    58
4-2 Future Work    59
Chapter. 5 References    60




                                

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