||Microfluidic systems for separation, counting, sorting, culture and differentiation of stem cells
||Department of Engineering Science
首先，第一個系統主要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。我們設計了一被動式微流體分選晶片，其中嵌有傾斜的屋簷式過濾器來將4-6 μm豐富的幹細胞篩選出來。利用緩衝液將細胞流體聚焦成一狹窄的流線後，之後進入傾斜式屋簷狀過濾器區，小顆粒慣性較小會隨著流體的路徑而通過孔徑為8 μm空隙，大顆粒則被阻擋下來而隨著往下流動的流體而帶走，來達到高分離效果。此屋簷式過濾器可以解決過濾大小細胞常見的阻塞問題。初步使用塑膠珠模擬實際狀況，結果可以得到約86 % 的分離效率。在分離羊水幹細胞實驗中可以達到良好的效率(82.8 %)，而重複其步驟可以將分離效率提高至97.1 %。
Microfluidic techniques have been recently developed for cell-based assays. In microfluidic systems, the objective is for these microenvironments to mimic in-vivo surroundings. With advantageous characteristics such as optical transparency and the capability for automating protocols, different types of cells can be cultured, screened and monitored in real time to systematically investigate their morphology and functions under well-controlled microenvironments in response to various stimuli. Recently, the study of stem cells using microfluidic platforms has attracted considerable interest. Even though stem cells have been studied extensively using bench-top systems, an understanding of their behavior in in-vivo-like microenvironments which stimulate stem cell proliferation and differentiation is still lacking. In this paper, several stem cell studies using microfluidic systems are purposed. The various miniature systems for stem cell separation/isolation, sorting, isolation, culture, differentiation, and stimulation, are then systematically introduced. Compared with conventional cell culture protocols, the microfluidic techniques provide versatile approaches to mimic more in vivo-like extracellular conditions for more realistic cell-based assay research. There still exist several inherent advantages including low biosamples/reagents consumption, a single integrated chip with multiple functions, and ability to run the array assays simultaneously. In this study, it was presented several new microfluidic devices fabricated based on SU-8 lithography process, a computer numerical controlled (CNC) milling for molds, and polydimethylsiloxane (PDMS) replica molding processes for stem cells researches.
First, a passive separation chip with louver-like structures in the microchannel is proposed as a filter to separate mesenchymal stem cells (MSCs) from amniotic fluid. Buffer solution is used to squeeze the sample flow by using the syringe pumps to form a narrow stream so that the sample flows close to the louver-like structures to obtain a higher separating efficiency. The device can alleviate the clogging problem and avoid the use of the external force such that cells will not be damaged during the separation process. Preliminary results show that the developed microfluidic device can perform a good separation of 86% (beads). It also shows that that the developed microfluidic device can perform a good separation of 82.8 % for MSCs. Furthermore, the separation process can be repeated to improve the separation efficiency to 97.1 %.
Another magnetic-bead technology integrated with the microfluidic system was purposed to develop a platform capable of isolating, counting, and sorting the hematopoietic stem cells. Since there is only an extremely small amount of stem cells existing in the umbilical cord blood, it is crucial to isolate and count the cell sample. In this research, the processes including mixing, transporting, counting and sorting can be completed automatically using the microfluidic control module. The target stem cells will be first captured by the antibody coated onto the magnetic beads, and then be successfully counted and sorted by a detection system.
In addition, a continuous microfluidic device capable of automating culturing and differentiating the MSCs was proposed. Microfluidic-based pneumatic trumpet-like micropump activated by two electromagnetic valves (EMVs) with three air chambers plus an elusive side-channel was used to suck the culture medium so that the medium in the culture area can be continuously supplied. Moreover, the waste can be moved through the elusive side-channel without contamination. The results represented that MSCs can be cultured and differentiated into different kinds of phenotypes stably for a long time. The stem cell culture chip not only can provide stable and well-defined microenvironments, but also features in low consumption of research resource.
Finally, an integrated microfluidic system capable of fine-tuning the insulin concentration automatically and applying different levels of shear stresses simultaneously was developed to investigate the effects of chemical and mechanical stresses on adipogenic differentiation of MSCs. It is comprised of a dilution device which can automatically fine-tune the concentrations of insulin for chemical stimulation on stem cells and three different levels of shear stresses produced by deflecting the PDMS membranes used to induce stem cells at the same time. The experimental results showed that an optimum insulin concentration of 10 μg/ml for differentiation of adipocytes can be determined. Moreover, the adipogenic differentiation can be suppressed by applying stronger shear stress and higher pulsation frequency of mechanical stimulation.
In summary, we have demonstrated several microfluidic based platforms of separation/isolation, counting, sorting, culture, differentiation and stimulation for the stem cell which may provide a promising development in the this new medical field.
List of table XI
List of figure XII
Chapter 1 Introduction 1
1.1 Microfluidics 1
1.2 Stem cells 3
1.2.1 Embryonic stem cells (ESCs) 3
1.2.2 Adult stem cells 4
1.2.3 Stem cell types and applications in microfluidics 7
1.3 Motivation and objectives 7
1.4 Scope and structure of the dissertation 8
Chapter 2 Theory 13
2.1 Fundamental of pneumatically-driven membrane-based actuators 13
2.2 The mechanism of the hormone 14
Chapter 3 Materials and Methods 16
3.1 Fabrication of microfluidic chips 16
3.1.1 Fabrication of masters using photolithography 16
3.1.2 Fabrication of masters using computer numerical control (CNC) milling 17
3.1.3 PDMS replication process 18
3.1.4 Chip packaging 18
3.2 Performances of microfluidic devices 18
3.2.1 Evaluation of the pumping rates 18
3.2.2 Experimental setups for fluidic control 19
3.2.3 Observation of the high-speed motions 19
3.3 Staining assay 20
3.3.1 Oil Red O staining 20
3.3.2 Alkaline phosphatase staining 20
Chapter 4 Development of the separation systems for stem cells 22
4.1 Introduction 22
4.2 Development of louver-array structures for separation of amniotic fluid mesenchymal stem cells 28
4.2.1 Design and Fabrication 28
184.108.40.206 Design 28
220.127.116.11 Fabrication 30
4.2.2 Experimental 30
18.104.22.168 Setup 30
22.214.171.124 MSCs immunofluorescence staining assay 31
126.96.36.199 Evaluation of the passive microfilter with louver-like structures 31
4.2.3 Results and discussions 32
188.8.131.52 Bead separation 32
184.108.40.206 Amniotic stem cell separation 35
4.2.4 Summary 36
4.3 Development of a integrated microfluidic system for isolation, counting and sorting of hematopoietic stem cells 37
4.3.1 Experimental 37
220.127.116.11 Procedure 37
18.104.22.168 Chip design 38
22.214.171.124 Fabrication process 40
126.96.36.199 Sample preparation 40
4.3.2 Result and discussion 41
188.8.131.52 Characterization of the microfluidic system 41
184.108.40.206 Cell isolation 43
220.127.116.11.1 Isolation of simulated cell samples 43
18.104.22.168.2 HSCs isolation 44
22.214.171.124 Cell detection 45
4.3.3 Summary 47
Chapter 5 Development of a continuous culture and differentiation platform for mesenchymal stem cells 62
5.1 Introduction 62
5.2 Design and fabrication 65
5.2.1 Design 65
5.2.2 Fabrication 66
5.3 Experimental 67
5.3.1 Setup 67
5.3.2 MSCs culture assay 68
5.3.3 MSCs differentiation assay 69
5.4 Result and discussion 71
5.4.1 Characterization of the microfluidic system 71
5.4.2 Culture and differentiation of AFMSCs 72
5.4.3 Immunofluorescence staining for AFMSCs 74
5.5 Summary 74
Chapter 6 Chapter 6 Development of a mechanical and chemical stimulation platform for mesenchymal stem cells 84
6.1 Introduction 84
6.2 Design and fabrication 88
6.2.1 Chip design 88
6.2.2 Fabrication 90
6.3 Experimental 90
6.3.1 Setup 90
6.3.2 MSCs culture and differentiation assay 91
126.96.36.199 MSCs culture assay 91
188.8.131.52 MSCs differentiation assay 92
184.108.40.206 Measurement of the differentiated adipocyte expression 92
6.4 Result and discussion 93
6.4.1 Characterization of the microfluidic system 93
220.127.116.11 Characterization of micropumps 93
18.104.22.168 Performance of the dilution device 94
6.4.2 Chemical stimulation of MSCs 95
6.4.3 Mechanical stimulation of MSCs 98
6.5 Summary 102
Chapter 7 Conclusions and Future Work 112
7.1 Discussion and Conclusions 112
7.2 Future Work 114
Curriculum Vitae 139
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