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研究生:Myagmartsend Enkhbat
研究生(外文):Myagmartsend Enkhbat
論文名稱:人類非小細胞肺癌細胞在顆粒單層膜表面的行為研究
論文名稱(外文):Modulation of Non-small Cell Lung Cancer A549 Cell Behavior Using Monolayer Colloidal Crystals (CCs)
指導教授:王鵬元
指導教授(外文):Peng-Yuan Wang
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:生醫材料暨組織工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:英文
論文頁數:84
中文關鍵詞:Non-small cell lung cancercolloidal crystalsdrug screening
外文關鍵詞:Non-small cell lung cancercolloidal crystalsdrug screening
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Background: In the last decades, researchers studied micro/nanoscale topographic cues on modulation of cancer cell behavior. Drug candidate and toxicity screening processes currently rely on results from early-stage in vitro cell-based assays expected to faithfully represent aspects of in vivo pharmacology and cancer treatment strategy. Occasionally new chemical or biological entities fail late-stage of human drug testing or refuse from regulatory even remove from the market for safety reasons after regulatory approvals due to unsatisfactory and inexpensive, high-throughput screening in vitro platform. Therefore, development of reliable translational assay toolkit for pharmacology and personalized medicine is the demand of this decade. In addition, generation of inexpensive, high-throughput screening in vitro platform with satisfactory in vivo prediction values would allow for a significant decrease in the number of animals used during the drug development process.

Aim: To fabricate a wide range of surfaces using monolayer colloidal crystals (CCs) for cancer cell culture. To investigate and screen the behavior of A549 non-small cell lung cancer cell on various CCs. To explore different drug responses of A549 cells using CCs based in vitro system.

Materials and Methods: Monolayer of CCs were fabricated based on evaporation induced confined area assembly (EICAA). CCs are composed of two or more types of particles including silicon, polystyrene, and polymethyl methacrylate. We designed a library of 24 CCs including binary CCs, mixed CC, and ternary CCs. We screened cancer cell behavior on different CCs and 5 binary CCs selected for drug screening study. A549 cell was selected for cell behavior study and drug screening experiment. Cisplatin, Doxorubicin were selected for drug screening. CCs were characterized using an optical microscope, scanning electron microscope, and sessile drop method for wettability. A549 cell behavior determined optical microscope, scanning electron microscope (SEM), MTT assay, CCK-8 assay, fluorescence staining and drug exposure. Quantitative analysis was performed using ImageJ and GraphPad Instat 3.0.

Results: Total 24 CCs have been made and half of them are high quality for long-term cell culture. A549 cells on BCCs showed lower cell density and smaller cell spreading area compared to TCPS. For example, cells on surface #4 (5SiPS0.4) have 496 ± 120 um2 spreading area compared with 1870 ± 695 um2 on TCPS at day 2. Cells on different CCs showed different cell density. For example, surface #8 (5SiPSC0.4) was the highest, while surface #6 (5SiPS0.1) was the lowest at day 4. From SEM images, cells show less filopodia, which means cell surface interactions reduced. Two drug were tested on CCs and TCPS for 24h and 72h.

Conclusion: We successfully fabricated CCs composed of different sizes and materials. These substrates investigated as next generation of cell culture tool to study the behavior of cancer cells and high-throughput screening. CCs substrates offer a wide range of properties for cell culture study.

Keywords: Non-small cell lung cancer, colloidal crystals, drug screening, cell viability
Background: In the last decades, researchers studied micro/nanoscale topographic cues on modulation of cancer cell behavior. Drug candidate and toxicity screening processes currently rely on results from early-stage in vitro cell-based assays expected to faithfully represent aspects of in vivo pharmacology and cancer treatment strategy. Occasionally new chemical or biological entities fail late-stage of human drug testing or refuse from regulatory even remove from the market for safety reasons after regulatory approvals due to unsatisfactory and inexpensive, high-throughput screening in vitro platform. Therefore, development of reliable translational assay toolkit for pharmacology and personalized medicine is the demand of this decade. In addition, generation of inexpensive, high-throughput screening in vitro platform with satisfactory in vivo prediction values would allow for a significant decrease in the number of animals used during the drug development process.

Aim: To fabricate a wide range of surfaces using monolayer colloidal crystals (CCs) for cancer cell culture. To investigate and screen the behavior of A549 non-small cell lung cancer cell on various CCs. To explore different drug responses of A549 cells using CCs based in vitro system.

Materials and Methods: Monolayer of CCs were fabricated based on evaporation induced confined area assembly (EICAA). CCs are composed of two or more types of particles including silicon, polystyrene, and polymethyl methacrylate. We designed a library of 24 CCs including binary CCs, mixed CC, and ternary CCs. We screened cancer cell behavior on different CCs and 5 binary CCs selected for drug screening study. A549 cell was selected for cell behavior study and drug screening experiment. Cisplatin, Doxorubicin were selected for drug screening. CCs were characterized using an optical microscope, scanning electron microscope, and sessile drop method for wettability. A549 cell behavior determined optical microscope, scanning electron microscope (SEM), MTT assay, CCK-8 assay, fluorescence staining and drug exposure. Quantitative analysis was performed using ImageJ and GraphPad Instat 3.0.

Results: Total 24 CCs have been made and half of them are high quality for long-term cell culture. A549 cells on BCCs showed lower cell density and smaller cell spreading area compared to TCPS. For example, cells on surface #4 (5SiPS0.4) have 496 ± 120 um2 spreading area compared with 1870 ± 695 um2 on TCPS at day 2. Cells on different CCs showed different cell density. For example, surface #8 (5SiPSC0.4) was the highest, while surface #6 (5SiPS0.1) was the lowest at day 4. From SEM images, cells show less filopodia, which means cell surface interactions reduced. Two drug were tested on CCs and TCPS for 24h and 72h.

Conclusion: We successfully fabricated CCs composed of different sizes and materials. These substrates investigated as next generation of cell culture tool to study the behavior of cancer cells and high-throughput screening. CCs substrates offer a wide range of properties for cell culture study.

Keywords: Non-small cell lung cancer, colloidal crystals, drug screening, cell viability
Acknowledgments.........I
Abstract.........II
Table of Contents.........IV
List of Figures.........VI
List of Tables.........VIII
Abbreviation List.........IX
Chapter 1. Introduction.........1
1.1 What is Cancer?.........1
1.2 Personalized Medicine.........1
1.3 Nanotechnology in Medicine.........2
1.4 In vitro Culture of Cancer Cell Lines.........6
Chapter 2. Literature Review.........7
2.1 Non-small Cell Lung Cancer.........7
2.2 In vitro Cell-Based Drug Screening.........7
Chapter 3. Monolayer Colloidal Crystals.........10
3.1 Introduction.........10
3.2 Experimental Section.........10
3.2.1 Materials.........10
3.2.2. Preparation of Various Colloidal Crystal Monolayers.........11
3.2.3. Substrate and Particle Characterization.........14
3.3 Results and Discussions.........14
3.3.1 Monolayer Colloidal Crystals (CCs) Fabrication and Characterization.........14
Chapter 4. Modulation of A549 Cell Behavior on CCs.........24
4.1 Introduction.........24
4.2 Experimental Section.........24
4.2.1 Materials.........24
4.2.2 Cell Line and Cell Culture.........25
4.2.3 Cell Viability Test on BCCs.........25
4.2.4 Immunofluorescence Staining of A549 Cells on CCs.........26
4.2.5 Drug Response of A549 Cell on Different BCCs.........26
4.2.6 Quantitative Analysis.........27
4.3 Results and Discussions.........27
4.3.1 Non-small Cell Lung Cancer (A549) Cell Culture on CCs.........27
4.3.2 Non-small Cell Lung Cancer (A549) Cell Culture on BCCs.........28
4.3.4 Drug Screening on Non-small Cell Lung Cancer (A549) Cells on BCCs.........29
Chapter 5. Capturing and Expansion of CTCs Using nanostructured surfaces.........58
Abstract
5.1 Introduction.........58
5.2 Capturing and Expansion of Tumor Cells.........59
5.3 Current Models for Tumor Research.........64
5.4 Conclusions.........65
Chapter 6. Modulation of PEI-Mediated.........67
6.1 Introduction.........67
6.2 Experimental Section.........67
6.2.1 Materials.........67
6.2.2 Fabrication of Binary Colloidal Crystals.........68
6.2.3 Silane PEG NH2 modification on BCCs.........68
6.2.4 Culture of A549 Cell 68
6.2.5 Gene Transfection and Fluorescent Staining.........69
6.2.6 Statistical Analysis.........69
6.3 Results and Discussions.........70
6.3.1 Gene Transfection Efficiency.........70
6.3.2 Silane PEG NH2 modification on BCCs.........70
Chapter 7. Conclusions.........74
Chapter 8. Future Works.........75
References.........76


List of Figures
Figure 2.1 Extracellular matrix factors affecting cell function in vivo.........9
Figure 3.1 Schematic illustration of fabrication of colloidal crystals.........15
Figure 3.2 Optical images of large surface areas displaying colloidal crystals structure.........16
Figure 3.3 SEM images of monolayer of colloidal crystal (CCs) heated at 93°C for 2 hours..........18
Figure 3.4 SEM images of monolayer of colloidal crystal (CCs) heated at 200°C for 1 min..........20
Figure 3.5 Water contact angles.........22
Figure 3.6 Zeta potentials of the colloidal particles.........23
Figure 4.1 Merged fluorescence images of A549 cells on CCs after 48h culture.........30
Figure 4.2 Merged fluorescence images of A549 cells on CCs after 96 h culture.........31
Figure 4.3 The mean cell density (cell nucleus counting).........32
Figure 4.4 Cell spreading area (single cell area) on CCs after 48 h culture.........33
Figure 4.5 BF and fluorescence images of A549 cells on BCCs and TCPS after 24 h culture.........34
Figure 4.6 BF and fluorescence images of A549 cells on BCCs and TCPS after 72h culture.........35
Figure 4.7 MTT assay. After 24h and 72h culture.........36
Figure 4.8 Mean cell density Measured from cells on BCCs and TCPS after 24 h and 72 h culture.........37
Figure 4.9 Viability per cell (OD value/Cell density) after 24 h and 72 h culture.........38
Figure 4.10 Cell spreading area (single cell area).........39
Figure 4.11 SEM images of A549 cells on BCCs after 24 h culture.........40
Figure 4.12 SEM images of A549 cells on BCCs and TCPS after 24h culture..........41
Figure 4.13 BF images of A549 cells on BCCs and TCPS after 24 h various concentration of doxorubicin exposure.........42
Figure 4.14 BF images of A549 cells on BCCs and TCPS after 72 h various concentration of doxorubicin exposure.........43
Figure 4.15 Fluorescence images of A549 cells on BCCs and TCPS after 24 h increasing concentration of doxorubicin exposure.........44
Figure 4.16 Fluorescence images of A549 cells on BCCs and TCPS after 72 h increasing concentration of doxorubicin exposure.........45
Figure 4.17 CCK-8 assay.........46
Figure 4.18 CCK-8 assay.........47
Figure 4.19 CCK-8 assay.........48
Figure 4.20 Mean cell density (cell nucleus counting).........49
Figure 4.21 Mean cell density (cell nucleus counting).........50
Figure 4.22 Viability per cell (OD value/Cell density) after 24 h (A) and 72 h (B) various concentration of DOX exposure.........51
Figure 4.23 Cell spreading area (single cell area) after 24 h (A) and 72 h (B) various concentration of DOX exposure.........52
Figure 4.24 BF images of A549 cells on BCCs and TCPS after 24h various concentration of cisplatin exposure.........53
Figure 4.25 BF images of A549 cells on BCCs and TCPS after 72 h various concentration of cisplatin exposure.........54
Figure 4.26 MTT assay.........55
Figure 4.27 MTT assay.........56
Figure 4.28 MTT assay.........57
Figure 5.1 Examples of nanostructured surfaces for CTC capturing.........60
Figure 5.2 A chaotic mixing channel is composed of two functional components.........61
Figure 5.3 NanoVelcro cell-affinity substrates.........62
Figure 5.4 Nanofiber matrix used for (A) selective cell capturing and (B) 3D cell culture..........63
Figure 5.5 Schematic of the micropillar device with GO- modified magnetic nanoparticles for CTC capturing [68]..........64
Figure 6.1 Fluorescence images of A549 cells after 24 h, 48 h and 72 h of transfection.........71
Figure 6.2 Mean cell density (cell nucleus counting).........72

List of Tables
Table 1.1 Clinically approved nanoparticles for anti-cancer therapeutic applications.........4
Table 3.1 List of colloidal crystals.........11
Table 3.2 Combination list of colloidal crystals.........13
Table 5.1 Example of nanostructured surfaces for capturing tumor cells.........60
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