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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 范士岡(Shih-Kang Fan) | |
dc.contributor.author | Kuan-Lun Ho | en |
dc.contributor.author | 何寬倫 | zh_TW |
dc.date.accessioned | 2021-06-17T06:06:01Z | - |
dc.date.available | 2024-01-21 | |
dc.date.copyright | 2019-01-21 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2019-01-16 | |
dc.identifier.citation | 參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71666 | - |
dc.description.abstract | 微流道系統、3D列印等微流體技術,發展至今已經相當成熟並被應用於工程、材料以及生物醫學等領域。然而,提及同時操控液體與固體粒子的能力,使用現有的系統與技術仍無法實際達成。在本研究中,我們首先運用電控微流體(Electromicrofluidic platform, EMF)平台以介電濕潤(Electrowetting on dielectric, EWOD)與液體介電泳(Liquid dielectrophoresis, LDEP)驅動不同種類水膠預聚物,並以粒子介電泳(Dielectrophoresis, DEP)排列水膠預聚物中的聚苯乙烯粒子,建構圖案化的微結構,成功展現電控微流平台跨尺度操控的能力。接著,我們將此平台與技術應用於生物領域,依據不同的細胞類型,給予相對應成長微環境,同時藉由電控微流體平台整合不同交聯策略的生物相容性水膠,建構以水膠為基底的仿生細胞培養支架,進行多種細胞的共培養後形成具有適當的細胞表型與功能的體外肝臟小葉組織。而本研究所使用的水膠材料分別為光交聯的Gelatin methacryloyl (GelMA)與Polyethylene glycol diacrylate (PEGDA)、化學交聯的纖維蛋白凝膠(Fibrin gel)以及熱交聯的基底膜基質(Matrigel)。細胞株分別為人類肝癌細胞(Human hepatocellular carcinoma, Huh-7)、人類臍帶靜脈內皮細胞(Human umbilical vein endothelial cells, HUVEC)以及人類單核球細胞(Human monocytic cells, THP-1)。而在仿生組織的培養過程中,我們將觀察細胞的生長以及細胞與細胞之間的交互作用,最後施加會導致肝毒性產生的藥物 (Acetaminophen, APAP),並量測肝臟細胞白蛋白的分泌改變量與細胞的活性變化,成功地將電控微流體排台應用於體外仿生組織的建構。此外,這個體外仿生肝臟組織可以作為基礎醫學研究、不同種類藥物測試甚至是病原體研究的理想體外模型。在未來,電控微流體平台同時跨尺度操控液體與固體的能力將能夠被應用於其他研究甚至是生醫、材料工程等領域,具極大的開發條件與潛力。 | zh_TW |
dc.description.abstract | In this study, electromicrofluidic (EMF) platform integrating electrowetting-on-dielectric (EWOD) and dielectrophoresis (DEP) forces is used to pattern multiple hydrogel prepolymers containing solid particles into in-vivo-like hepatic lobule arrangements. Biocompatible hydrogel with different concentration and stiffness was manipulated by the electromicrofluidic platform to construct a hydrogel-based scaffold for cell culture and form an in vitro liver tissue with proper cell morphology and phenotypic function, and our goal is to engineer a liver lobule tissue with blood vessel formation. Therefore, four kinds of hydrogels were used in this research, including photo-crosslinkable hydrogel, gelatin methacryloyl (GelMA) and polyethylene glycol diacrylate (PEGDA), chemically-crosslinkable hydrogel, fibrin gel, and thermally-crosslinkable hydrogel, matrigel. On the other hand, human hepatocellular carcinoma (Huh-7), human umbilical vein endothelial cells (HUVEC) and differentiated human monocytic cells, THP-1 were cultured in biocompatible hydrogels to form the in-vitro biomimetic liver tissue. To construct in vitro hepatic-lobule-like microstructures, Huh-7 and HUVEC mixed with THP-1 were dispersed in GelMA prepolymers respectively with appropriate concentrations. Furthermore, we measured the albumin secretion from different culture conditions and evaluated the function of our liver lobule tissue. For the applications, we investigated the influence of drug hepatotoxicity on liver function. At last, to improve the cell proliferation of HUVEC, we have cultured HUVEC in different extracellular matrixes, expecting that the microvascular formation of endothelial cells may improve the function of our liver tissue. In the future, this engineered tissue can serve as an ideal in vitro model for basic medical research, drug screening and pathogen interaction. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T06:06:01Z (GMT). No. of bitstreams: 1 ntu-107-R05522115-1.pdf: 66166635 bytes, checksum: dda079dee4833347c0b058e57fc27cd3 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 目錄
致謝 i 中文摘要 ii Abstract iii 第一章 緒論 1 1-1 研究背景 1 1-2 文獻回顧 2 1-2-1 組織工程與體外仿生組織 2 1-2-2 細胞外基質-水膠材料 4 1-2-3 二維及三維細胞培養 6 1-2-4 細胞共培養 7 1-2-5 細胞與微環境的排列 9 1-2-6 體外肝臟仿生組織 12 1-3研究方法與目的 24 第二章 電控微流體技術簡介 26 2-1 介電濕潤理論 26 2-2 介電泳理論 28 2-2-1 液體介電泳 28 2-2-2 粒子介電泳 30 2-3 電控微流體平台相關應用 31 第三章 實驗系統與材料介紹 34 3-1 電控微流體晶片製程 34 3-1-1 電控微流體晶片介紹與選擇 34 3-1-2 清洗氧化銦錫玻璃基板 34 3-1-3 旋轉塗佈正光阻 35 3-1-4 圖案化光阻: 曝光、顯影、定影 35 3-1-5 濕蝕刻氧化銦錫導電層 36 3-1-6 旋轉塗佈介電層 36 3-1-7 旋轉塗佈疏水層 37 3-1-8 上板製程 37 3-2 電控微流體平台系統 38 3-2-1 控制系統 38 3-2-2 電控微流體平台 39 3-2-3 電極圖案設計 40 3-3 細胞培養系統與相關實驗材料 41 3-3-1 操作及細胞培養設備 41 3-3-2 細胞來源與培養 42 3-3-3水膠材料 46 3-3-4 螢光染劑與螢光顆粒 50 第四章 實驗與結果討論 51 4-1 單頻率訊號跨尺度水膠與粒子之操控 51 4-2 複合頻率訊號跨尺度操控異質水膠與粒子 56 4-3 藥物毒性對於肝臟細胞之影響 61 4-4 建構三維仿生肝臟組織 65 4-5 藉由複合頻率排列細胞建構三維仿生肝臟組織 75 4-2 血管內皮細胞於不同種類水膠培養 81 第五章 實驗結論與未來展望 89 5-1 實驗結論 89 5-2 未來展望 91 參考文獻 92 圖目錄 圖 1組織工程概念示意圖[14]。 3 圖 2以組織工程的方法建構出體外仿生模型[1]。 3 圖 3以不同種類水膠進行細胞的二維、三維培養,探討細胞的生長情形[18]。 4 圖 4藉由控制濃度改變水膠物理性質並分析細胞生長型態[25]。 5 圖 5上皮細胞於體內以及體外三維環境生長型態比較[28]。 6 圖 6圖案化共培養肝臟細胞與纖維母細胞形成藥物開發之平台[4]。 7 圖 7利用細胞共培養的方式刺激血管內皮細胞生長[30]。 8 圖 8以水膠包覆細胞進行培養,探討物理性邊界對細胞生長的影響[38]。 9 圖 9利用表面聲波技術調整頻率來達到排列細胞的目的[41]。 10 圖 10組織結構與排列對於細胞功能性影響之研究[44]。 11 圖 11體外肝臟仿生組織研究之建構技術與其應用分類圖[9]。 12 圖 12以介電泳技術產生不均勻電場來排列多種細胞,建構出肝臟小葉組織[48]。 13 圖 13以微製程技術建立肝竇模型,並以微結構設計模仿內皮細胞的功能[49]。 14 圖 14以微製程技術建立肝臟小葉模型進行細胞共培養,作為肝毒性藥物與不同種藥物之間的互相影響的測試平台[53]。 15 圖 15利用動態培養系統建構體外肝細胞分區模型(Liver zonation model) [54]。 16 圖 16利用生物列印技術,建構體外三維仿生肝臟組織應用於藥物毒性的測試[61]。 17 圖 17利用光顯影技術搭配水膠材料進行多種細胞的共培養,建構體外仿生肝臟小葉組織,並探討二維培養、三維培養以及三維細胞共培養對肝臟細胞特有蛋白質分泌量的影響[62]。 18 圖 18利用電控微流體技術排列含有多種細胞的水膠,建構體外仿生肝臟小葉組織,探討探討藥物之間的互相作用與藥物肝毒性的分析[66]。 19 圖 19體外仿生肝臟小葉組織結構設計圖。(比例尺: 1 mm) 24 圖 20固著液滴任意兩相界面上的表面張力分布圖。 26 圖 21施加電壓前後電荷累積於電濕潤與介電濕潤結構之情形[73]。 27 圖 22介電泳力與毛細管內液體之靜力平衡狀態,呈現當初H. Pellat原始的實驗模型[77]。 28 圖 23以平行電極板裝置利用液體介電泳力驅動液體[78]。 29 圖 24在不均勻電場中驅動粒子的正負介電泳力[80]。 30 圖 25整合介電濕潤與粒子介電泳於電控微流體平台上,達到跨尺度的操控液體與細胞和粒子[75]。 31 圖 26利用電控微流體平台產生液體介電泳力,建構出不需實體流道壁的虛擬微流道[78]。 32 圖 27利用電控微流體平台建構體外異質三維細胞培養微結構[42]。 33 圖 28電控微流體平台系統示意圖。 38 圖 29彙整三種不同電控微流體技術於電控微流體平台上。 (a)電控微流體平台側視圖。 (b)電控微流體平台上視圖。 (c)介電泳技術示意圖。 39 圖 30以肝臟小葉結構為基礎所設計之電極圖案。(比例尺: 1 mm) 40 圖 31 Gelatin與Methacrykic anhydride反應後合成GelMA之化學反應圖[83]。 47 圖 32光起始劑照射特定波段之UV光後產生自由基,而與GelMA進行交聯形成水膠[83]。 48 圖 33光起始劑照射特定波段之UV光後產生自由基,而與PEGDA進行交聯形成水膠[84]。 48 圖 34人類肝臟小葉組織構造圖[87]。 52 圖 35利用電控微流體平台跨尺度操控不同分子量之PEGDA水膠並同時排列PEGDA 575中之6 μm聚苯乙烯粒子。(比例尺: 1 mm) 52 圖 36含有螢光染劑之三維水膠微結構分佈圖。 53 圖 37三維水膠微結構於螢光顯微鏡下觀察之結果。(比例尺: 500 μm) 53 圖 38三維水膠微結構於共軛焦顯微鏡下觀察之結果。 54 圖 39三維水膠微結構於掃描電子顯微鏡下觀察之結果。 55 圖 40利用電控微流體平台跨尺度操控5% GelMA水膠與PEGDA水膠並同時排列水膠中的聚苯乙烯粒子。(比例尺: 1 mm) 57 圖 41含有螢光染劑之三維水膠與螢光粒子微結構分佈圖。 57 圖 42三維異質水膠微結構於螢光顯微鏡下觀察之結果。(比例尺: 500 μm) 58 圖 43三維異質水膠微結構於掃描電子顯微鏡下觀察之結果。 59 圖 44乙醯胺酚(acetaminophen)代謝途徑圖。 61 圖 45藥物毒性對於肝臟細胞影響實驗時間對照圖。 63 圖 46不同APAP藥物濃度對於肝臟細胞白蛋白分泌量之影響,結果為第13天與第10天量測到之數據的比值。 64 圖 47不同APAP藥物濃度對於肝臟細胞白蛋白分泌量之影響,結果為第13天與第12天量測到之數據的比值。 64 圖 48以電控微流體技術排列含有Huh-7細胞的5% GelMA以及含有HUVEC和THP-1細胞的10% GelMA,形成仿生肝臟小葉細胞培養微結構。 66 圖 49三維仿生人類肝臟小葉組織構造圖。 66 圖 50三維細胞共培養處理流程與方式示意圖。 67 圖 51 Huh-7、HUVEC以及THP-1共培養於圖案化水膠結構之培養結果。 67 圖 52三維仿生組織於螢光顯微鏡下觀察之結果。(比例尺: 500 μm) 68 圖 53三維仿生組織於共軛焦顯微鏡下觀察之結果。 69 圖 54體外肝臟細胞不同培養條件下白蛋白分泌曲線。 70 圖 55細胞培養與藥物施加之時間軸。 71 圖 56具肝毒性藥物對於不同細胞培養條件下細胞活性之影響。 72 圖 57具肝毒性藥物對於不同細胞培養條件下,施加藥物前後對於肝臟細胞白蛋白分泌之影響。 73 圖 58比較具肝毒性藥物對於不同細胞培養條件白蛋白分泌量之影響,結果為第13天與第10天量測到之數據的比值。 74 圖 59以電控微流體平台驅動含有HUVEC與THP-1細胞的3% GelMA水膠,並同時藉由複合頻率排列5% GelMA水膠中的Huh-7細胞,形成更仿生的肝臟小葉組織。(比例尺: 1 mm) 76 圖 60三維仿生人類肝臟小葉組織細胞與水膠結構分佈圖。 76 圖 61三維細胞共培養處理流程與方式示意圖。 77 圖 62藉由優化三維仿生組織之處理方式,來維持曝光後細胞於光交聯水膠內之活力(cell viability),進而控制多組樣品細胞生長較為一致,以利後續培養與藥物測試之條件。此為仿生組織培養2天後之結果。(比例尺: 1 mm) 78 圖 63血管內皮細胞於濃度較低之3% GelMA水膠中培養2天後有明顯之增生情形。(比例尺: 50 μm) 78 圖 64圖案化排列水膠與肝臟細胞之仿生肝臟小葉組織培養數天後之生長情形。 80 圖 65血管內皮細胞於5% GelMA水膠環境中單獨培養結果。(比例尺: 50 μm) 81 圖 66血管內皮細胞於基板上進行二維生長之型態。(比例尺: 50 μm) 82 圖 67血管內皮細胞之空泡(Vacuole)結構,於細胞培養後12 h所發現。 83 圖 68培養血管內皮細胞於三維纖維蛋白凝膠培養結果。 83 圖 69以共軛焦顯微鏡拍攝血管內皮細胞於三維水膠中培養4天之生長狀態。(比例尺: 50 μm) 84 圖 70分別單獨培養血管內皮細胞與共培養血管內皮細胞和分化單核球細胞,觀察7天後的結果。(比例尺: 50 μm) 85 圖 71血管內皮細胞以及分化後之單核球細胞共培養之培養結果,內皮細胞呈血管管狀型態生長,而分化後之單核球細胞呈球狀之型態生長。 87 圖 72培養第六天後發現分化後之單核球細胞穿過血管內皮細胞行成的血管管壁,於管腔中持續生長,並參考相關文獻以佐證之[96]。(比例尺: 50 μm) 87 圖 73以共軛焦顯微鏡拍攝三維血管內皮細胞與分化後之單核球細胞共培養之生長狀態。(比例尺: 50 μm) 88 表目錄 表 1體外仿生肝臟模型綜合比較。 20 表 2體外仿生肝臟模型分析方式比較表。 21 表 3利用電控微流體平台建構三維圖案化微結構之實驗參數。 51 表 4利用電控微流體平台建構異質三維圖案化微結構之實驗參數。 60 表 5藥物毒性對於肝臟細胞影響之實驗參數。 62 表 6利用電控微流體平台建構三維仿生肝臟小葉組織之實驗參數。 65 表 7利用電控微流體平台建構三維仿生肝臟小葉組織之實驗參數。 75 表 8以纖維蛋白凝膠混和基底膜基質共培養血管內皮細胞與分化後之單核球細胞實驗參數。 86 | |
dc.language.iso | zh-TW | |
dc.title | 電控微流體平台跨尺度操控顆粒與水膠並建構體外肝臟小葉組織 | zh_TW |
dc.title | Particles and hydrogels manipulation to construct hepatic lobule tissue on electromicrofluidic platform | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 劉承賢(Cheng-Hsien Liu),許聿翔(Yu-Hsiang Hsu),張維典(Wei-Tien Chang) | |
dc.subject.keyword | 電控微流體,跨尺度操控,異質水膠微結構,仿生肝臟小葉組織,微血管結構,藥物測試平台, | zh_TW |
dc.subject.keyword | electromicrofluidics,cross-scale manipulation,heterogeneous microstructures,liver lobule tissue,microvascular,drug testing platform, | en |
dc.relation.page | 99 | |
dc.identifier.doi | 10.6342/NTU201900088 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-01-16 | |
dc.contributor.author-college | 工學院 | zh_TW |
dc.contributor.author-dept | 機械工程學研究所 | zh_TW |
顯示於系所單位: | 機械工程學系 |
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