進階搜尋


   電子論文尚未授權公開,紙本請查館藏目錄
(※如查詢不到或館藏狀況顯示「閉架不公開」,表示該本論文不在書庫,無法取用。)
系統識別號 U0026-2308201822195800
論文名稱(中文) 簡單製造大小可控之微孔用於培養均勻體外多細胞腫瘤球狀體與開發新的治療方式
論文名稱(英文) Simple fabrication of size-controlled microwells for the generation of uniform in vitro multicellular tumor spheroids and discovery of novel therapeutics
校院名稱 成功大學
系所名稱(中) 生物醫學工程學系
系所名稱(英) Department of BioMedical Engineering
學年度 106
學期 2
出版年 107
研究生(中文) 邱巧宜
研究生(英文) Chiao-Yi Chiu
電子信箱 judyq0401@gmail.com
學號 P86051040
學位類別 碩士
語文別 英文
論文頁數 59頁
口試委員 指導教授-涂庭源
口試委員-張憲彰
口試委員-王仰高
口試委員-陳盈元
中文關鍵字 多細胞腫瘤球體  微孔  肝癌  艾黴素  光熱治療 
英文關鍵字 multicellular tumor spheroid  microwell  liver cancer  doxorubicin  photothermal treatment 
學科別分類
中文摘要 肝細胞癌是最常見的原發性肝臟惡性腫瘤,因為早期診斷和治療尚未充分發展,因此難以預測。三維(three-dimensional, 3D)細胞培養與傳統二維(two-dimensional, 2D)相比涵蓋許多結構和功能已被證明是一種更具仿生的工具。而在3D體外細胞培養中,多細胞腫瘤球體(multicellular tumor spheroid, MTS)的培養已逐漸被接受為一種可模擬部分體內組織的模型,它重現了體內生物學功能和藥物開發的幾種關鍵病理特徵。在生成MTS的許多現有平台中,微孔(microwell)技術通過定義微結構之幾何用於培養尺寸可控的3D MTS,適用於後續接種之不同實驗功能測定,從而成為體外3D培養的優異平台。本論文提出了一種簡易實惠的微孔製造方法,主旨在使此一方法能方便的結合大多數生物和醫學實驗室的傳統工作流程,用於培養體外3D MTS。這項研究包括三個主要目的:(1)使用傳統的CO2雷射雕刻機進行尺寸控制microwell的快速成型,並通過調節microwell原型的參數來控制可變尺寸以形成肝癌體外3D MTS; (2)結合microwell與常規多孔板概念證明高通量藥物篩選的可行性; (3)通過對刀豆素A(Concanavalin A, ConA) 修飾的二氧化矽—碳空心球 (silica-carbon hollow spheres, SCHSs) 進行光熱處理來探索新的腫瘤治療手段。
結果顯示,microwell的尺寸可控於400 µm到700 µm之間。在microwell系統中利用對肝癌細胞Huh-7進行了長達5天的培養,可形成3D MTS尺寸為250 µm至520 µm,並呈現出良好的細胞活力和形狀。此技術也具備整合於高通量藥測的流程架構,並透過直接加工microwell於傳統的96孔板盤進行演示。2D條件下的IC50濃度為9.3 µM,相對於兩種3D條件0 mm、10 W(M0@10)和 -3 mm,15 W(M-3@15)的microwell中,IC50濃度分別為42.8和52.3 µM。光熱治療結果顯示濃度於500:200 µg/mL的Con A與 SCHSs在孵育2小時後可結合達至最佳濃度。藉由活死染色細胞測定法(live-dead cell assay)計算細胞相對活性,進一步驗證MTS暴露於3 W/cm2之近紅外光雷射照射20分鐘後,細胞活性之相對螢光比率相較於未照射之MTS降低8倍,證實了光熱治療作為潛在治療干預的可行性。
因此,可以預期此本論文之microwell平台技術,將有機會透過產生肝癌體外3D MTS,作為開發癌症相關具功效之治療的體外平台。
英文摘要 Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. It has a poor prognosis because it is often diagnosed in the advanced stage when treatments are limited. Three-dimensional (3D) cell culture models have become powerful tools in cancer research, as they better simulate the in vivo physiological microenvironment than traditional 2D cell cultures. Tumor cells cultured in a 3D system as multicellular tumor spheroids (MTS) recapitulate several critical in vivo characteristics that allow the study of biological functions and drug discovery. Microwell technology is best platform for generating MTS as it provides geometrically defined microstructures for culturing size-controlled MTS amenable for various downstream functional assays.
This thesis presents a simple and economical microwell fabrication methodology that be conveniently incorporated into the conventional workflow used to generate MTS. This study had three main objectives: (1) To perform rapid prototyping of size-controlled microwells using a conventional CO2 laser engraver, and to control the variable sizes by fine-tuning the parameters of microwell prototyping to generate hepatic MTS; (2) To combine microwell technology with conventional multi-well plate-based cell culture methods for proof-of-concept of high-throughput drug screening; (3) To explore novel therapeutic interventions through photothermal treatment of concanavalin A (ConA)-modified silica–carbon hollow spheres (SCHSs).
The microwells were 400–700 µm in diameter, and hepatic MTS cultured in them for up to 5 days grew to 250–520 µm with good viability and shape. To demonstrate the ability to integrate the microwell fabrication with a high-throughput workflow using the conventional multi-well plate system, a conventional 96-well plate was employed for proof-of-concept drug screening. The half maximal inhibitory concentrations of doxorubicin were determined to be 9.3 µM in 2D conditions and 42.8 and 52.3 µM in both 3D conditions, namely microwells fabricated at focal lengths and laser powers of 0 mm and 10 W, and -3 mm and 15 W, respectively.
The optimal concentration for ConA binding to SCHSs was 500:200 µg/mL after a 2 h incubation to best bind with MTS. Based on this concentration for further photothermal treatment, the live/dead cell viability assay assessed the relative cell viability through exposure to 3 W/cm2 near-infrared laser for 20 min. The relative fluorescence intensity showed an eight-fold reduction in cell viability, confirming the feasibility of photothermal treatment as a potential therapeutic intervention. In conclusion, using the microwell platform to generate MTS may be an effective tool for discovering therapeutic modalities for cancer treatment.
論文目次 致謝 I
中文摘要 II
Abstract IV
Table of Contents VI
List of Figures IX
List of Abbreviations XI
Chapter 1 Introduction 1
1.1 Background 1
1.2 Research Aims 4
1.3 Literature review 6
1.3.1 MTSs 6
1.3.2 Platforms for generating MTSs 9
1.3.3 Drug screening and photothermal therapy 12
1.4 Thesis overview 15
Chapter 2 Materials and Methods 17
2.1 Microwell prototyping 17
2.2 SEM 18
2.3 Cell culture 19
2.4 Formation of MTS 20
2.4.1 Laser-ablated microwells 20
2.4.2 Hanging drop 22
2.5 MTS morphology and viability analysis 23
2.6 Immunofluorescence 25
2.7 2D and 3D drug screening 26
2.8 Workflow of photothermal therapy 28
Chapter 3 Results and Discussion 30
3.1 Fabrication of Microwells 30
3.2 Generation of MTS 36
3.3 2D and MTS high-throughput anticancer drug screening 41
3.4 Photo-thermal therapy 44
3.4.1 Binding capacity of ConA-SCHSs to MTS 44
3.4.2 Photo-thermal treatment through bound ConA-SCHSs 50
Chapter 4 Conclusions 53
Chapter 5 Future works 55
Reference 56
參考文獻 [1] R.Siegel et al., “Cancer statistics , 2015” CA Cancer J Clin, 2015.
[2] M. J.Thun et al., “The global burden of cancer: Priorities for prevention” Carcinogenesis, 2009.
[3] J.Balogh et al., “Hepatocellular carcinoma: a review,” J. Hepatocell. Carcinoma, vol. Volume 3, pp. 41–53, 2016.
[4] X.Xu et al., “Three-dimensional in vitro tumor models for cancer research and drug evaluation” Biotechnology Advances. 2014.
[5] Y. C.Chen et al., “The photothermal effect of silica-carbon hollow sphere-concanavalin A on liver cancer cells” J. Mater. Chem. B, vol. 3, no. 12, pp. 2447–2454, 2015.
[6] X.Gong et al., “Generation of multicellular tumor spheroids with microwell-based agarose scaffolds for drug testing” PLoS One, 2015.
[7] G.Mehta et al., “Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy” J. Control. Release, vol. 164, no. 2, pp. 192–204, Dec.2012.
[8] M.Eiraku et al., “Self-organizing optic-cup morphogenesis in three-dimensional culture” Nature, 2011.
[9] M. P.VShekhar et al., “Drug resistance: challenges to effective therapy” Curr. Cancer Drug Targets, vol. 11, no. 5, pp. 613–623, 2011.
[10] F.Andre et al., “Implication of tumor microenvironment in the resistance to chemotherapy in breast cancer patients” Current Opinion in Oncology. 2010.
[11] S. R.Kim et al., “Expression of Keratin 10 in Rat Organ Surface Primo-vascular Tissues” JAMS J. Acupunct. Meridian Stud., vol. 4, no. 2, pp. 102–106, 2011.
[12] F.Hirschhaeuser et al., “Multicellular tumor spheroids: An underestimated tool is catching up again” J. Biotechnol., 2010.
[13] L. J.Nelson et al., “Low-shear modelled microgravity environment maintains morphology and differentiated functionality of primary porcine hepatocyte cultures” Cells Tissues Organs, 2010.
[14] A.Takai et al., “Three-dimensional Organotypic Culture Models of Human Hepatocellular Carcinoma” Sci. Rep., 2016.
[15] R. M.Sutherland et al., “A Multi-component Radiation Survival Curve Using an in Vitro Tumour Model” Int. J. Radiat. Biol. Relat. Stud. Physics, Chem. Med., vol. 18, no. 5, pp. 491–495, Jan.1970.
[16] C.Hsiao et al., “Microwell regulation of pluripotent stem cell self-renewal and differentiation” vol. 2, no. 4, pp. 266–276, 2013.
[17] N.Ono et al., “Stirring apparatus for cell culture” Bellco Glas. Inc., no. 19, 1982.
[18] M.Vinci et al., “Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation” BMC Biol., 2012.
[19] D.DelDuca et al., “Spheroid preparation from hanging drops: Characterization of a model of brain tumor invasion” J. Neurooncol., 2004.
[20] C.Fischbach et al., “Engineering tumors with 3D scaffolds” Nat. Methods, 2007.
[21] D.Kloß et al., “Drug testing on 3D in vitro tissues trapped on a microcavity chip” Lab Chip, 2008.
[22] Y. Y.Choi et al., “Controlled-size embryoid body formation in concave microwell arrays” Biomaterials, 2010.
[23] P.Zorlutuna et al., “Microfabricated biomaterials for engineering 3D tissues” Advanced Materials. 2012.
[24] E. J.Vrij et al., “3D high throughput screening and profiling of embryoid bodies in thermoformed microwell plates” Lab Chip, 2016.
[25] A. M.Bratt-Leal et al., “Incorporation of biomaterials in multicellular aggregates modulates pluripotent stem cell differentiation” Biomaterials, 2011.
[26] J Comley “Rapidly Becoming a Preferred” Drug Discov., 2017.
[27] S.Kong et al., “Calcium phosphate porous sintered body and production thereof” vol. 2, no. 12, pp. 12–15, 2011.
[28] G. N.Hortobagyi et al., “Immediate and long-term toxicity of adjuvant chemotherapy regimens containing doxorubicin in trials at M.D. Anderson Hospital and Tumor Institute” NCI Monogr. a Publ. Natl. Cancer Inst., 1986.
[29] J.Cox et al., “Mechanisms of doxorubicin resistance in hepatocellular carcinoma” Author Manuscr., vol. 3, no. 1, pp. 57–59, 2016.
[30] K. M.Tewey et al., “Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II” Science, 1984.
[31] B.Liu et al., “Antiproliferative activity and apoptosis-inducing mechanism of Concanavalin A on human melanoma A375 cells” Arch. Biochem. Biophys., 2009.
[32] R.Weissleder et al., “A clearer vision for in vivo imaging” Nat. Biotechnol., 2001.
[33] T. Y.Tu et al., “Rapid prototyping of concave microwells for the formation of 3D multicellular cancer aggregates for drug screening” Adv. Healthc. Mater., 2014.
[34] H. L.LaMarca et al., “Three-dimensional growth of extravillous cytotrophoblasts promotes differentiation and invasion” Placenta, 2005.
[35] K.Yokobori et al., “Intracellular localization of pregnane X receptor in HepG2 cells cultured by the hanging drop method,” Drug Metab. Pharmacokinet., 2017.
[36] J.Saarikangas et al., “Regulation of the Actin Cytoskeleton-Plasma Membrane Interplay by Phosphoinositides” Physiol. Rev., 2010.
[37] J. A.Tunggal et al., “E-cadherin is essential for in vivo epidermal barrier function by regulating tight junctions” EMBO J., 2005.
[38] C.Lovit et al., “Advanced Cell Culture Techniques for Cancer Drug Discovery” Biology (Basel)., 2014.
[39] S. K.Lam et al., “Lectins: Production and practical applications” Applied Microbiology and Biotechnology. 2011.
[40] L. L.Fu et al., “Plant lectins: Targeting programmed cell death pathways as antitumor agents” International Journal of Biochemistry and Cell Biology. 2011.
[41] G. M.Edelman et al., “The Covalent and Three-Dimensional Structure of Concanavalin A” Proc. Natl. Acad. Sci., 1972.
[42] K.Welsher et al., “Deep-tissue anatomical imaging of mice using carbon nanotube fluorophores in the second near-infrared window” Proc. Natl. Acad. Sci., 2011.
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2021-08-31起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2021-08-31起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw