||Development of microfluidic systems for micro-scale animal cell culture- from cell separation, microencapsulation, micro-dispensing to perfusion 3-dimensional cell culture
||Department of Engineering Science
3-D cell culture
Perfusion cell culture
以細胞為基礎之生醫研究已被廣泛運用於生命科學領域中探討細胞生理反應與環境因子之關係。傳統細胞操控之項目步驟主要包含了細胞分離、純化，固定包埋，流體定量傳輸以及細胞培養。然而這些操作方法大多無法運用在處理小量的生物樣本體積量。甚且，傳統細胞培養過程中需要消耗大量資源(例如：細胞數量)，進而影響了細胞基礎分析研究結果之生產率。此外，傳統的細胞培養方式無法提供具有生理意義、均質、且穩定之細胞培養環境。然而，近幾年來，由於微流體技術被驗證出許多優勢能夠改善傳統技術之缺點，且藉由其本身微小尺度之物理意義不同於傳統大尺度，微流體技術能夠提供許多獨特之機能是為傳統技術無法達到。因此，在此篇論文中提出了許多由微流體技術建構之高效率細胞處理方式以及高通量化之細胞培養方法。其先利用微雕機或者微影技術製作母模結構，再以聚二甲基矽氧烷 (PDMS: poly-dimethylsiloxane) 翻模建置出所需晶片。首先，一微流體技術建構之微型過濾晶片被提出用於微量細胞或是微珠之分離。其分離機制是利用可撓性的薄膜，加以適當氣體壓力驅此可撓性的薄膜產生不同下壓程度而產生不同大小的通道以用於篩選不同大小之細胞或微珠。甚且，利用整合於晶片當中之微型幫浦以達成自動化流體流向控制以及提供一機制防止於分離步驟當中之阻塞現象。接著，提出一新型的海藻膠微珠成型微流體裝置並且應用於細胞包埋。此裝置整合了氣動式振動原件且其下方連接著針頭，因此當針頭振動時，變會驅使於海藻膠液滴產生於油相當中，再利用重力方式，讓海藻膠液滴沉入氯化鈣溶液中固化形成海藻膠微珠。而不同的海藻膠微珠大小可藉由適當調控海藻膠流速以及氣動式振動原件振動頻率搭配而達成。此外，一微流體建構之微型取樣器被提出能夠送樣樣品量小於微升體積量。其特色便是整合氣壓驅動之薄膜當作快速吸取/送樣定量液體之原件。藉由適當控制氣壓驅動之薄膜元件當中之壓力釋放時間，便能夠快速取樣且送量不同量之樣品量範圍從0.05-0.45微升 (最小樣品量單位為0.05微升)。因此，此裝置提供了一易整合之多樣品量取樣送樣裝置。甚且，一新式微流體建置之連續式、微型三維細胞培養晶片亦被提出並應用於高通量生物分析研究。其主要特色為整合了利用新型C形狀微型幫浦建構之高通量培養液傳輸系統具有防止液體回流、高通量細胞膠體注入之功能。藉由此細胞培養晶片微型化之特色，其不僅提供了一生理意義、均質、且穩定之細胞培養環境，更大幅減少了細胞培養時所需之生物樣品消耗量。這些特點皆高度適用於高通量化以及高精確之三維細胞培養系統。最後，為了證實此研究所提出之裝置，將所提出之微流體裝置被應用於分離/純化、包埋關節軟骨細胞，以及探討關節軟骨細胞與其生長環境中酸鹼度之影響。實驗結果表示微型過濾晶片能提供高效率之體積量受限之細胞，其分離篩選率(93%)且不會對細胞造成傷害(細胞存活率：96%)之操作。而被分離出的關節軟骨細胞更進一步利用之海藻膠微珠成型裝置完成將細胞包埋於海藻膠微珠之程序，同樣的，此裝置亦不會對細胞造成傷害死亡於操作過程當中(細胞存活率：94±2%)。此外，微型取樣器亦成功用於調配出不同酸鹼值之培養液並將調配完之培養液與關節軟骨細胞一同放入新式細胞培養晶片培養以及分析最後細胞代謝產物。甚且為了比較微型化連續式細胞培養以及傳統細胞培養之差異，實驗結果更與一般大型靜置式的細胞培養結果有所比較。因此，此研究中所提出的微流體裝置，不僅提供了簡單、自動化、容易控制、條件均一、不傷害細胞生理、低汙染之細胞操作以及培養方法，更提供了高通量以及與生物體相似的培養環境以利細胞生理研究之進行。
Cell-based assays have been widely utilized in life science-related area to quantitatively investigate the link between the cellular responses and the tested conditions for decades. Conventional cell handling techniques mainly involve the cell isolation, separation, immobilization, liquid dispensing, and cell culture practice. These operations, however, might not be able to deal well with the biological sample with a small size. In addition, the commonly-used cell culture protocols might consume more experimental research resources (e.g. number of cells), and therefore the throughput of a cell-based assay might be compromised. More importantly, traditional cell cultures could not provide a stable, well-defined, and physiologically-meaningful culture conditions for cell-based assays due to the design of cell culture format. During the past decade, there have been tremendous advances in microfluidics. Due to the significant differences in several physical phenomena between microscale and macroscale devices, microfluidic technology provides unique functionality, which is not previously possible by using traditional techniques. This study reports several new microfluidic devices for high-performance cell handling and for high-throughput cell culture. All these devices fabricated based on a computer numerical controlled (CNC) milling or SU-8 lithography process for molds and polydimethylsiloxane (PDMS) replica molding processes. Firstly, to achieve cell isolation and separation, a new microfluidic-based filter was presented. The filtration separation mechanism is based on the pneumatically tunable deformation of PDMS membranes, which block the fluid channel with a varied degree. This defines the dimensions of the remaining passageway of fluid channel and thus the passage of the microbeads/cells with a specific size. Because of the miniaturization and tunable characteristics of separation performance, not only is the proposed device applicable to perform cell separation under the circumstance that either harvested specimen is limited to the cell content in a sample is sparse, but it also paves a new rout to separate/isolate cells in a simple, controllable and cell-friendly manner. To immobilize cells for 3-D cell culture purpose, a new microfluidic device for continuous generation of alginate microbeads was proposed. The working mechanism is based on the use of a pneumatically-driven vibrator to continuously spot tiny alginate microdroplets in a thin oil layer. The temporarily formed alginate microdroplets are soon sinking into a sterile calcium chloride solution to become gelled microbeads. By regulating the alginate suspension flow rate and the pulsation frequency of the integrated vibrator, the alginate microbeads can be produced in a size-controllable manner. Furthermore, a microfluidic-based pneumatically-driven micro-dispenser was demonstrated for precise pipetting of sub-microliter samples. The key feature of the micro-dispenser is the use of a suction membrane to provide a driving force for precise and quick aqueous liquid sampling and pipetting. The micro-dispenser features in the elegant control of the releasing time of the air pressure in the pneumatic chamber of the pressure-generating unit, contributing to precise pipetting of aqueous liquid volumes ranging from 0.05 μl to 0.45 μl (the minimum unit is 0.05 μl) achieving the multi-volume dispensing capability. By means of proper combinations, the liquid of various volumes would be easily sampled. In addition, a new perfusion-based, micro three-dimensional (3-D) cell culture platform was proposed for high-throughput bioassays using enabling microfluidic technologies. The main characteristics of the chip are the capability of multiple medium deliveries without any back-flow by using the new design pneumatic C-shape micropumps, and the function of efficient cells/hydrogel scaffold loading. Based on the inherent natures of miniaturized perfusion 3-D cell culture, the cell culture chip not only can provide stable, well-defined and more biologically-relevant culture environments, but also features in low consumption of research resource. All these traits are found particularly useful for high-precision and high-throughput 3-D cell culture-based assays. Finally, all the microfluidic devices proposed in the research were demonstrated to perform the process including separation, microencapsulation of the chondrocytes and investigation the effect of extracellular pH on chondrocyte functions. Experimental results showed that the chondrocytes from the limited enzymatically-digested tissue suspension can be successfully separated by using the microfluidic-based filter with an excellent cell separation efficiency of 93 % and a high cell viability of 96%. Moreover, the separated chondrocytes were encapsulated in alginate microbeads with high cell viability (94±2%) by using the microfluidic alginate microbead generator. Besides, a micro-scale perfusion 3-D cell culture-based assay to study the effect of extracellular pH on chondrocyte was successfully demonstrated using the proposed cell culture chip and the micro-dispenser was used to adjust the different pH value of the medium. The results were also compared with the same evaluation based on conventional static cell culture with larger culture scale. As a whole, these microfluidic systems proposed in the study provide a simple, automatic, controllable, uniform, cell friendly, less contaminated manner for cell manipulation and culturing and may facilitate a high-throughput cell culture based assay in the more in vivo-like environment.
List of Figures XII
Chapter 1: Introduction 1
1.1 Introduction to animal cell culture 1
1.2 Microfluidics as a niche technology for cell culture based assay 4
1.2.1 Introduction to microfluidics 4
1.2.2 Microfluidic-based Lab on a chip 5
1.3 Motivation and objectives 6
1.4 Scope and structure of the dissertation 7
Chapter 2 Theory 11
2.1 Chemical gradients in 3-D cell culture construct 11
2.2 Fundamentals of pneumatically-driven membrane-based actuators 13
2.2.1 Deflection of the circular-type PDMS membrane 13
2.2.2 Deflection of the rectangular-type PDMS membrane 14
2.2.3 Operation time of the pneumatic-based actuators 14
Chapter 3 Materials and Methods 18
3.1 Microfabrication of microfluidic devices 18
3.1.1 Fabrication of masters using photolithography 18
3.1.2 Fabrication of masters using computer numerical control (CNC) milling 19
3.1.3 PDMS replica molding 20
3.1.4 Chip packaging 20
3.1.5 PDMS surface modification 21
3.2 Performances of microfluidic devices 21
3.2.1 Evaluation of the pumping rates 21
3.2.2 Observation of the high-speed motions 22
3.2.3 Experimental setups for fluidic and temperature control 22
3.2.4 Image analysis 22
3.3 Bioassays 23
3.3.1 Cell number and cell viability 23
3.3.2 Lactate 23
3.3.3 Glycosaminoglycan 23
Chapter 4 Development of a tunable micro filter modulated by pneumatic pressure 26
4.1 Introduction 26
4.2 Design and Fabrication 28
4.2.1 Design 28
4.2.2 Fabrication 31
4.3 Evaluation of separation performances 32
4.3.1 Evaluation of the tunable membrane filter 32
4.3.2 Evaluation of the sorting ratio 32
4.3.3 Evaluation of the filtration flux rate 33
4.4 Results and discussion 33
4.4.1 Characterization of the pneumatically-tunable microfluidic filter 33
4.4.2 Separation performance 35
4.5 Summary 37
Chapter 5 Development of Pneumatically-driven micro-vibrators to generate alginate microbeads 46
5.1 Introduction 46
5.2 Design and Fabrication 48
5.2.1 Design 48
5.2.2 Fabrication 50
5.3 Experimental 51
5.3.1 Setup 51
5.3.2 Evaluation of flow output 51
5.3.3 Evaluation of the vertical displacement of the vibrators 52
5.3.4 Evaluation of alginate microbead size and uniformity 52
5.4 Results and discussion 53
5.4.1 Characterization of the pneumatically-driven micro-vibrators 53
5.4.2 Evaluation of the size and uniformity of alginate microbeads 55
5.5 Summary 56
Chapter 6 Development of pneumatically driven micro-dispenser for sub-micro-liter pipetting 64
6.1 Introduction 64
6.2 Design and Fabrication 65
6.2.1 Design 65
6.2.2 Fabrication 67
6.3 Experimental 67
6.3.1 Evaluation of the micro-dispenser 67
6.3.2 Evaluation of the titration ability of the micro-dispenser 68
6.4 Results and Discussion 68
6.4.1 Characterization of the micro-dispenser 68
6.4.2 Comparison the performance of the micro-dispenser and the commercial pipettor 70
6.5 Summary 71
Chapter 7 Development of high-throughput perfusion-based micro 3-D cell culture platform 78
7.1 introduction 78
7.2 Design and Fabrication 81
7.2.1 Design 81
7.2.2 Microfabrication 84
7.3 Experimental 84
7.3.1 Setup 84
7.3.2 Pumping action and performance 85
7.4 Results and Discussion 85
7.4.1 Cells/agarose loading mechanism 85
7.4.2 Pneumatic micropumps for multiplex culture medium delivery 86
7.5 Summary 87
Chapter 8 The separation, microencapsulation, cell culture of articular chondrocytes-demonstration for high throughput 3-D cell culture-based assay 94
8.1 Preparation of chondrocytes suspension 95
8.2 Separation of chondrocytes from enzymatically-digested cartilage tissue suspension by using the micro filter 95
8.3 Pneumatically-driven micro-vibrators to generate alginate microbeads for the microencapsulation of cells 97
8.4 Adjustment of the pH value of the culture medium by using the micro-dispenser 98
8.5 Investigation of the effect of extracellular pH on chondrocyte function using the microfluidic cell culture chip 99
8.6 Summary 102
Chapter 9 Conclusions and Future Work 107
9.1 Conclusions 107
9.2 Future Work 110
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