使用壓力輔助網路暨液珠累積系統以形成陣列式懸滴

簡易檢索 / 詳目顯示

回結果列表

研究生：	卓晉逸 Cho, Chin-Yi
論文名稱：	使用壓力輔助網路暨液珠累積系統以形成陣列式懸滴 Formation of Hanging Drop Arrays Using Pressure-Assisted Network for Droplet Accumulation System (PANDAS)
指導教授：	黃振煌 Huang, Jen-Huang
口試委員:	許佳賢 Hsu, Chia-Hsien 王潔 Wang, Jane
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：	2020
畢業學年度：	107
語文別：	英文
論文頁數：	63
中文關鍵詞：	微流體逐層建構、懸掛式細胞液珠培養、壓力控制系統、自動形成液滴陣列
外文關鍵詞：	Microfabrication technique, Hanging drop cell culture, Pressure control system, Automatic formation of droplets array
相關次數：	點閱：2 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

藥物篩選是一個十分長時間的過程，近年來，高通量篩選藥物成為了重要的技術，因為高通量篩選可以加速藥物研發的過程。三維度培養系統模擬適當的模型類似於體內腫瘤的微環境系統，在高通量篩選的要求下，由表面張力將細胞液滴支撐在板下側的懸掛式液珠方法是用於藥物開發的三維度形式的細胞球體的關鍵技術。透過手動使用定量吸管，可以將許多含有培養基混合細胞的液滴分配在培養板上，然而即便使用多通道的定量吸管，這種形成液珠的步驟也稱為勞動密集型程序；當液滴數接近數千時，不可能手動產生如此大量的液滴。最新的技術已經可以通過使用自動定量吸管機在懸滴培養板（例如：384孔盤）上注入細胞以達成高通量的開發，但是此種方法需要仰賴造價昂貴及占空間的機械手臂，因此用於液滴陣列的設施需要改良成小型和成本低廉的裝置。
在這項研究中，我們開發了一種壓力輔助網路暨液珠累積系統（PANDAS），它將多通道定量吸管技術和液滴培養技術結合到同一個系統中，並且無需使用其他裝置即可形成懸滴式細胞液陣列。 PANDAS可以通過微流體通道網絡施加負壓，使用戶能夠快速且自發地產生懸滴，從而實現液滴陣列的均勻分佈。可以通過修改設備板的孔來調整懸滴的大小。此外，PANDAS還可用於為多組織實驗並用於不同的細胞類型(例如：幹細胞培養)。此技術非常有潛力開發用於體外模型研究的高通量平台。

The 3D culturing system simulates an adequate model that displays a microenvironment similar to in vivo tumor. In the requirement of high throughput screening (HTS), hanging drop, a method that scatters droplets of the cell into the underside of the plate due to surface tension becomes a critical technique to form cell spheroids in a 3D format for drug development. By using a pipette, numerous of droplets that contain the medium and cells can be simply and manually dispensed on a culture plate. However, this pipetting step is known as a labor-intensive procedure even using a multi-channel pipette. When the droplet number comes to nearly thousands, it is impossible to generate such a large quantity of droplets manually. Recent technology has been developed by using an automated pipetting machine to introduce cells on hanging drop culture plates (e.g. 384 wells) to facilitate the HTS. Nevertheless, the facility for plating droplet arrays still requires additional space and cost-effective equipment.
In this study, we developed a Pressure-Assisted Network for Droplet Accumulation System (PANDAS) that combined both multi-pipetting and droplet culturing techniques into a single platform for the formation of hanging drop array without using external setup. The PANDAS can allow the users to generate hanging drops rapidly and spontaneously by applying a negative pressure through the microfluidic channel networks, enabling the uniform distribution of droplet array. The size of the hanging droplet can be tuned by modifying the device plate’s wells. Moreover, the PANDAS can be used to introduce different cell types for the multi-tissue experiment. The promising technology presented here enables a possibility to develop HTS platform for in vitro model research.

Abstract    i
Acknowledgements    iv
Contents    v
List of illustrations    vii
List of Table    ix
Chapter 1: Introduction and paper review    1
1-1 Introduction & paper review    1
1-1-1 Background & motivation    1
1-1-2 2D culture    2
1-1-3 3D culture    3
1-1-4 Spheroid cell culture    4
1-1-5 Drug screening    6
1-2 Hanging drop method    6
1-3 Innovation & purpose    9
Chapter 2. Experimental design & system fabrication    10
2-1 Device design    10
2-1-1 Principle    10
2-1-2 Design of 9-holes plate    11
2-1-3 Design of 25-holes & 100-holes plate    12
2-1-4 Design of 14-holes round plate    13
2-1-5 PET piece helps to suspend droplets    14
2-1-6 Sealing petri dish chamber    14
2-1-7 Removable plate connect with PDMS ring    15
2-1-8 Design of 8-holes round plate for drug screening    15
2-2 Fabrication of the device & material selection    16
2-2-1 Fabrication of the plate    16
2-2-2 Operation of CNC MDX-40A    19
2-2-3 Fabrication of the PET piece    21
2-2-4 Fabrication of PDMS ring    22
2-2-5 The fabrication of petri dish chamber    23
2-2-6 Middle-scale of the device    25
2-3 Pressure control by a syringe pump    25
2-3-1 Operation of the syringe pump    26
2-3-2 Operation of Differential pressure transmitter 984    27
2-4 Cell culture    30
2-4-1 Sterilization    31
2-4-2 A549 cells culture on the device    31
2-4-3 Control group: culture A549 cells on a petri dish by hanging drop method    32
2-5 Cytokines assay    32
2-5-1 Microscope    32
2-5-2 Live and dead staining    33
Chapter 3. Results and Discussion    35
3-1 Plate design evolution    35
3-1-1 Hydrophilic-based design    35
3-1-2 Prototypes of the current plate    36
3-1-3 Modification in the current method    38
3-1-4 Selection of the hole size    40
3-1-5 Real device structure measurement    41
3-2 Formation of droplets    43
3-2-1 Time consumption of formation    45
3-2-2 Successful rate    47
3-2-3 Formation pressure of the droplet    50
3-3 Cell culture    51
3-3-1 Comparison of cell numbers before & after the process    52
3-3-2 Hanging drop method for culturing other kinds of cells by PANDAS    54
3-3-3 Stability of droplets in an incubator    55
3-4 Drug screening of multiple droplets    56
3-4-1 Principle    56
3-4-2 Preliminary results    57
3-5 Conclusions    59
Chapter 4. Future work    61
4-1 Time-lapse photography    61
4-2 1000-holes plate    61
Chapter 5 Reference    62

                                

1. Rahib, L., et al., Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer Res, 2014. 74(11): p. 2913-21.
2. Imamura, Y., et al., Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer. Oncol Rep, 2015. 33(4): p. 1837-43.
3. Tung, Y.C., et al., High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. Analyst, 2011. 136(3): p. 473-8.
4. Misun, P.M., et al., Multi-analyte biosensor interface for real-time monitoring of 3D microtissue spheroids in hanging-drop networks. Microsystems & Nanoengineering, 2016. 2(1).
5. Francesco Pampaloni, E.G.R.a.E.H.K.S., The third dimension bridges the gap between cell culture and live tissue. nature reviews molecular cell biology, 2007. 8: p. 839-845.
6. Aijian, A.P. and R.L. Garrell, Digital microfluidics for automated hanging drop cell spheroid culture. J Lab Autom, 2015. 20(3): p. 283-95.
7. Yoshii, Y., et al., The use of nanoimprinted scaffolds as 3D culture models to facilitate spontaneous tumor cell migration and well-regulated spheroid formation. Biomaterials, 2011. 32(26): p. 6052-8.
8. Ahmed, E.M., Hydrogel: Preparation, characterization, and applications: A review. J Adv Res, 2015. 6(2): p. 105-21.
9. Singh, M., et al., Production of Uniform 3D Microtumors in Hydrogel Microwell Arrays for Measurement of Viability, Morphology, and Signaling Pathway Activation. Assay Drug Dev Technol, 2015. 13(9): p. 570-83.
10. Storch, K., et al., Three-dimensional cell growth confers radioresistance by chromatin density modification. Cancer Res, 2010. 70(10): p. 3925-34.
11. Liu, T., B. Lin, and J. Qin, Carcinoma-associated fibroblasts promoted tumor spheroid invasion on a microfluidic 3D co-culture device. Lab Chip, 2010. 10(13): p. 1671-7.
12. Bithi, S.S. and S.A. Vanapalli, Microfluidic cell isolation technology for drug testing of single tumor cells and their clusters. Sci Rep, 2017. 7: p. 41707.
13. Wang, W., et al., 3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells. Biomaterials, 2009. 30(14): p. 2705-15.
14. Onder, T.T., et al., Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res, 2008. 68(10): p. 3645-54.
15. HAMMOND, T.G.H.A.J.M., Optimized suspension culture: the rotating-wall vessel. Physiology-Renal Physiology, 2001. 281(1): p. F12-F25.
16. Amann, A., et al., Development of an innovative 3D cell culture system to study tumour--stroma interactions in non-small cell lung cancer cells. PLoS One, 2014. 9(3): p. e92511.
17. Hsiao, A.Y., et al., 384 hanging drop arrays give excellent Z-factors and allow versatile formation of co-culture spheroids. Biotechnol Bioeng, 2012. 109(5): p. 1293-304.
18. Kunz-Schughart, L.A., et al., The use of 3-D cultures for high-throughput screening: the multicellular spheroid model. J Biomol Screen, 2004. 9(4): p. 273-85.
19. Juergen Friedrich, C.S., Reinhard Ebner & Leoni A Kunz-Schughart, Spheroid-based drug screen: considerations and practical approach. nature protocols, 2009. 4: p. 309-324.
20. Sutherland, R.M., Cell and environment interactions in tumor microregions: the multicell spheroid model. Science, 1988. 240(4849): p. 177-184.
21. Ivascu, A. and M. Kubbies, Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. J Biomol Screen, 2006. 11(8): p. 922-32.
22. FRANZBLAU, L.A.C.A.S.G., Microplate Alamar Blue Assay versus BACTEC 460 System for High-Throughput Screening of Compounds against Mycobacterium tuberculosis and Mycobacterium avium. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 1997. 41: p. 1004–1009.
23. Tronser, T., et al., Droplet microarray: miniaturized platform for rapid formation and high-throughput screening of embryoid bodies. Lab Chip, 2018. 18(15): p. 2257-2269.
24. Nielsen, N.E.T.K., Generation of Multicellular Tumor Spheroids by the Hanging-Drop Method. Tissue Engineering, 2007: p. 141-151.
25. Wu, H.W., et al., A PDMS-Based Microfluidic Hanging Drop Chip for Embryoid Body Formation. Molecules, 2016. 21(7).
26. Frey, O., et al., Reconfigurable microfluidic hanging drop network for multi-tissue interaction and analysis. Nat Commun, 2014. 5: p. 4250.
27. Kelm, J.M., et al., Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng, 2003. 83(2): p. 173-80.

簡易檢索 / 詳目顯示

相關論文