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系統識別號 U0002-1008200900001200
中文論文名稱 以明膠微圖案進行細胞之操控貼附、培養與監控
英文論文名稱 Application of Gelatin Micropatterns in Cell Attachment, Culture and In-Situ Monitoring
校院名稱 淡江大學
系所名稱(中) 機械與機電工程學系博士班
系所名稱(英) Department of Mechanical and Electro-Mechanical Engineering
學年度 97
學期 2
出版年 98
研究生中文姓名 歐育誠
研究生英文姓名 Yu-Cheng Ou
學號 693340068
學位類別 博士
語文別 中文
口試日期 2009-07-07
論文頁數 166頁
口試委員 指導教授-楊龍杰
委員-陳建源
委員-施文彬
委員-黃吉宏
委員-康尚文
委員-楊龍杰
中文關鍵字 戊二醛  明膠  交聯  細胞培養  癌細胞  阻抗感測  即時監控 
英文關鍵字 glutaraldehyde  gelatin  crosslink  cell culture  tumor cell  impedance sensing  in-situ monitoring 
學科別分類
中文摘要 本文提出一種新穎戊二醛交聯明膠之微成型技術,並且利用所成型之明膠微圖案來吸引癌細胞的生長貼附,除了以加入感光增感劑曝照紫外光(ultra-violet)交聯明膠方式與用戊二醛選擇性交聯方式之外,進一步利用氧氣電漿(O2 plasma)蝕刻方式來製作明膠微圖案,本新穎之明膠微圖案成型方式可以確保明膠之交聯程度、避免以戊二醛選擇性交聯方式所產生的過度交聯(over-crosslink)、增加明膠微圖案與玻璃基材之間的黏著程度,線寬解析度最佳可到達2微米,而利用傳統光蝕刻微影技術(photolithography)可以成功的製作出戊二醛交聯明膠之微圖案,利用明膠材料之特性,成功的吸引癌細胞產生選擇性之貼附生長。
控制細胞生長的位置對於生醫感測器的製作、組織工程、生醫電子以及基礎生物研究而言是相當重要的,而本文則提供一個用以控制細胞生長位置的材料,也進一步將此材料實際應用於生醫感測器,監控細胞生長貼附狀態。在細胞培養過程中,細胞的黏著生長(cell adhesion)及其蔓延(cell spreading)是黏著型細胞的基本生長過程,其生長過程關係著人體的防禦系統、組織的形成與訊息傳遞路徑。而在個體細胞層級中,細胞的黏著生長(cell adhesion)與蔓延(cell spreading)關聯著單一細胞的許多特性,而單一細胞形態的改變,傳統上則使用光學的方式來進行量測,然而生醫感測器則提供了另一種量測方式。長久以來,電子細胞基質阻抗判斷技術(Electric Cell-substrate Impedance Sensing, ECIS)已經被認定是有效的方法,利用ECIS技術可以監測黏著型細胞之形態、活力以及細胞周遭環境變化。
本文結合了ECIS技術、明膠微圖案成型技術與光學方式來製作一新穎之細胞培養系統,此系統包含一個具有即時觀察光學模組之培養箱以及整合明膠微圖案之ECIS感測晶片,此光學模組可以用於即時觀察培養箱中不同活體細胞目標,除了可透過光學模組觀察活體細胞活動,進行影像監控之外,接近細胞大小之小尺明膠微圖案直接被成型於工作電極上方,藉此明膠微圖案使細胞產生選擇性黏著,利用明膠微圖案控制細胞生長於感測電極上方,進一步透過感測晶片輸出之阻抗訊號變化,監控細胞生長貼附之狀態,同時透過光學觀察模組之即時影像也可確認量訊號變化來自細胞之不同生長貼附狀態。
英文摘要 This work proposes a novel technique to fabricate micropatterns of glutaraldehyde (GA)-crosslinked gelatin and induce the attachment of tumor cells to the gelatin micropatterns. It provides another method to crosslink gelatin other than using the photo-sensitizing agents or selective GA-crosslinked technique. The gelatin micropatterns are fabricated by O2 plasma etching process. This novel technique can ensure the degree of crosslink, prevent the over-crosslink from pattern deformation and enhance the adhesion between the gelatin and glass slide. The best spatial resolution of the gelatin micropatterns can be reached to 2μm. The micropatterns of GA-crosslinked gelatin can still be made successfully by the conventional photolithography. By the properties of gelatin, tumor cells are successfully attracted to produce a selective.
It is important to control living cells onto the given surface for biosensor fabricating, tissue engineering, bioelectronics and basic biology studies. This thesis herein proposes a material to control cell positioning on the surface. Moreover, the material is actually employed in biosensor to monitor morphology of living cells. During the cell culture process, cell adhesion and cell spreading are fundamental processes of adherent cells. Furthermore, cell adhesion and cell spreading are also crucial to signal transduction pathways of a cell, the formation of tissues and the body’s defense system. At the individual cell level, some properties of a living cell are associated with cell adhesion and cell spreading processes. Traditionally, the changes of individual cell morphology are measured using the optical methods. In addition to the optical methods used to monitor the cell morphology of living cells, biosensors provide another means to monitor the changes of cell morphology. ECIS methods have long been regarded as a valid approach to monitor the morphology, viability, and environmental change of the adherent cells.
This thesis combines ECIS technique, fabrication of gelatin micropattern and optical methods to set up a new cell culture system. This system includes a culture incubator with a compact optical real-time monitoring module and an ECIS sensor chip with gelatin micro patterns. The optical observing module is used for observing different targets of living cells in the incubator. Furthermore, the optical module can be used to monitor the cell behavior. In the meantime, the gelatin micropatterns fabricated firmly on the ECIS working electrodes have a small size which is purposely comparable to cells. By the gelatin micropatterns, cells are attracted to produce a selective adhesion during the stages of falling and attachment of cell culture. By this way, living cells can be controlled onto the given working electrode surface. The morphology of living cell can be monitored by changes of impedance signal from ECIS chip. Meanwhile, the data attributed to various cell morphologies also can be confirmed by the real-time image of the optical module.
論文目次 目錄
中文摘要...........................................................................I
英文摘要...........................................................................III
目錄...................................................................................V
圖目錄...............................................................................VIII
表目錄...............................................................................XV
第一章 緒論 ..................................................................1
1-1 微機電系統...............................................................1
1-2 動物細胞培養...........................................................5
1-3 研究動機...................................................................7
1-4 文獻回顧...................................................................9
1-5 研究目的...................................................................13
1-6 文章架構...................................................................15
第二章 明膠製程材料與明膠圖案製程.....................17
2-1 明膠製程材料簡介..................................................18
2-1-1 明膠材料(gelatin)..................................................18
2-1-2 重鉻酸鉀(K2Cr2O7).............................................21
2-1-3 戊二醛交聯劑(glutaraldehyde)............................22
2-2 明膠微圖案製程.....................................................22
2-2-1 感光明膠成型明膠微圖案.................................22
2-2-2 戊二醛選擇性交聯成型明膠微圖案................25
2-2-3 氧氣電漿蝕刻成型戊二醛交聯明膠微圖案...36
2-3 三種明膠微成型技術之比較................................46
2-4 與其他蛋白質或膠原微圖案成型技術之比較...47
第三章 明膠微圖案應用於細胞培養.........................49
3-1 細胞無菌培養操作技術建立................................49
3-1-1 無菌操作基本技術..............................................52
3-1-2 細胞培養液之調配..............................................52
3-1-3 細胞解凍與培養..................................................55
3-1-4 細胞計數(cell counting).......................................60
3-1-5 細胞繼代培養(subculture)...................................63
3-1-6 細胞冷凍保存......................................................65
3-2 交聯明膠薄膜表面接觸角量測...........................68
3-3 交聯明膠微圖案之培養液態環境安定性...........71
3-4 明膠微圖案之細胞選擇性生長...........................72
3-4-1 明膠微圖案之細胞培養測試............................72
3-4-2 不同明膠微圖案對細胞生長之影響................76
3-4-3 明膠微圖案誘導單一細胞之黏著生長............79
第四章 細胞培養影像監視模組之設計與製作.........82
4-1 具即時觀察拍攝之CO2培養箱..........................82
4-2 具即時觀察拍攝CCD模組之設計與製作.........83
4-3 具即時觀察拍攝CCD模組之特點及功效.........92
第五章 整合明膠微圖案之電阻抗感測晶片.............94
5-1 電阻抗式感測晶片之可行性評估與試作............94
5-2 電阻抗式感測晶片設計........................................98
5-3 電阻抗式感測晶片製作........................................100
5-3-1 氧化銦錫(ITO)感測電極之製作........................100
5-3-2 成型誘導細胞貼附之明膠微圖案.....................113
5-3-3 訊號連接電路與PDMS矽膠環之製作............116
第六章 電阻抗感測晶片之訊號量測.........................129
6-1 量測實驗設備架設說明........................................129
6-2 感測晶片工作特性量測........................................134
6-3 細胞貼附生長於感測晶片後之現地量測............137
第七章 結論與未來建議.............................................145
7-1 本文貢獻之彙整....................................................145
7-2 未來建議................................................................150
參考文獻.......................................................................153
論文著述目錄...............................................................162
附錄A...........................................................................164
附錄B...........................................................................165
圖目錄
圖1-1 以明膠為阻擋層作微壓印轉印蛋白質圖形 ............... 9
圖1-2 以PDMS微型印章轉印蛋白質製作細胞微圖案 ............. 9
圖1-3 以機器手臂做快速大量蛋白質點印刷 .................. 10
圖1-4 以金屬薄膜自我組裝操控細胞生長位置 .................................. 10
圖1-5 CMOS多電極陣列電路晶片 ....................................................... 11
圖1-6 封裝上PDMS高分子之CMOS多電極陣列晶片 ...................... 11
圖1-7 ECIS技術量測單一細胞貼附過程示意圖 .................................. 12
圖1-8 阻抗式感測電極上視圖 .............................................................. 13
圖1-9 ECIS技術量測架設示意 .............................................................. 13
圖1-10 論文架構之樹狀圖 .................................................................... 16
圖2-1 膠原蛋白分子鏈結構 ............................. 18
圖2-2 (a)明膠單體結構;(b)明膠結構鏈 ................. 19
圖2-3 明膠中所含氨基酸成份 ........................... 19
圖2-4 明膠分子鏈結構 ............................. 19
圖2-5 明膠溶液濃度與融點關係圖 ...................................................... 20
圖2-6 高分子光架橋反應 ...................................................................... 21
圖2-7 試藥級重鉻酸鉀粉末 .................................................................. 21
圖2-8 感光明膠製作明膠微圖案製作流程圖 ...................................... 23
圖2-9 感光明膠補強聚對二甲苯結構示意圖 ...................................... 24
圖2-10 感光明膠補強聚對二甲苯空腔微結構 .................................... 24
圖2-11購自SIGMA之試藥級明膠粉末 ................................................ 25
圖2-12 明膠粉末吸水膨潤後 ................................................................ 25
圖2-13 隔水加熱溶解明膠粉末 ............................................................ 26
圖2-14 戊二醛選擇性交聯成型明膠微圖案製作流程圖 .................... 28
圖2-15 光阻殘留於明膠微圖案上方 .................................................... 29
圖2-16 曝光後產生光阻脫層之試片(左下) ........................................ 29
圖2-17 表面無光阻殘留之明膠微圖案 ................................................ 30
圖2-18 產生皺紋之明膠結構表面輪廓掃描圖 .................................... 30
圖2-19 戊二醛與明膠交聯反應機制 .................................................... 33
圖2-20 交聯時間過長之交聯明膠微圖案 ............................................ 33
圖2-21 過度交聯明膠微圖案之表面輪廓.............................................. 34
圖2-22 不同戊二醛濃度在不同交聯時間所產生之過交聯距離 ........ 34
圖2-23 適當交聯時間控制無毛邊明膠微圖案 .................................... 36
圖2-24 氧氣電漿蝕刻戊二醛交聯明膠微圖案製程 ............................ 37
圖2-25 完成製作之明膠微圖案 ............................................................ 40
圖2-26 電子顯微鏡拍攝氧氣電漿蝕刻成型明膠微圖案 .................... 41
圖2-27 原子力顯微鏡掃描量測明膠微圖案厚度 ................................ 42
圖3-1 無菌操作台氣流示意圖 .............................................................. 50
圖3-2 本實驗室細胞培養機台:CO2培養箱、無菌操作台 ................... 51
圖3-3 桌上型滅菌釜 .............................................................................. 51
圖3-4 細胞培養用之25T-flask培養瓶.................................................... 58
圖3-5 水浴槽37℃回溫 .......................................................................... 58
圖3-6 無菌操作 ...................................................................................... 58
圖3-7 放置培養瓶於培養箱內 .............................................................. 59
圖3-8 以倒立式相位差顯微鏡觀察細胞貼附狀況 .............................. 59
圖3-9 長滿培養瓶底部之HeLa子宮頸癌細胞 .................................... 59
圖3-10 血球計數盤 ................................................................................ 62
圖3-11 手按計數器 ................................................................................ 62
圖3-12 拍打flask底部使細胞脫離......................................................... 62
圖3-13以微量滴管來進行調製細胞冷凍液 .......................................... 67
圖3-14將細胞液由flask移入50mL離心管中........................................ 67
圖3-15 以吸唧管調配冷凍液 ................................................................ 67
圖3-16 以食指尖輕拍離心管底部分散細胞 ........................................ 68
圖3-17 加入冷凍細胞液於1mL冷凍小管 ............................................ 68
圖3-18 表面接觸角量測儀 .................................................................... 70
圖3-19 材料表面接觸角量測 ................................................................ 70
圖3-20 明膠試片連續浸泡培養液之腫脹測試 .................................... 72
圖3-21 全明膠薄膜細胞培養測試片 .................................................... 73
圖3-22 全明膠薄膜細胞培養測試片...................................................... 74
圖3-23 明膠微圖案細胞生長選擇性測試 ............................................ 76
圖3-24 HepG2癌細胞貼附生長於明膠微圖案 ..................................... 78
圖3-25 HeLa癌細胞貼附生長於明膠微圖案 ........................................ 78
圖3-26 NS-1癌細胞貼附生長於明膠微圖案 ........................................ 79
圖3-27 SEM拍攝HeLa細胞生長於不同尺寸明膠微圖案 ................... 80
圖3-28 SEM拍攝HepG2細胞生長於蜂窩形明膠微圖案 .................... 81
圖4-1 具即時觀察拍攝之CO2培養箱架構 ........................................... 83
圖4-2 相容於培養箱之光學觀察子系統3D示意圖 ............................ 83
圖4-3 微分干涉差(DIC)光路示意圖 ..................................................... 85
圖4-4 簡易DIC光路架設與成像測試.................................................... 87
圖4-5 氣密罩及微分干涉差之光機結構設計示意圖 .......................... 87
圖4-6 氣密罩及光機結構組合圖............................................................ 88
圖4-7 光機結構與實際光路對照圖 ...................................................... 88
圖4-8 光機結構零件爆炸圖 .................................................................. 89
圖4-9 實際微分干涉差之光機結構 ...................................................... 89
圖4-10 光機結構與氣密罩組裝置入細胞培養箱實體圖 .................... 90
圖4-11 即時拍攝HepG2癌細胞於明膠微圖案上方之活動 ................ 91
圖5-1 製作完成之感測電極 .................................................................. 95
圖5-2 無細胞系統下之循環伏安量測 .................................................. 97
圖5-3 活體細胞培養之循環伏安量測 .................................................. 97
圖5-4 ITO電阻抗感測電極尺寸設計示意圖 ........................................ 99
圖5-5 結合PDMS矽膠環之感測晶片示意圖 ...................................... 99
圖5-6 ITO感測電極製程流程圖 .......................................................... 101
圖5-7 完成黃金對準記號製作之ITO玻璃 ......................................... 102
圖5-8 金屬舉離法程序示意圖 ............................................................ 104
圖5-9 試作之梳狀ITO電極 ................................................................. 105
圖5-10 相位差顯微鏡觀察光線穿透ITO效果 ................................... 105
圖5-11 不同時間之ITO蝕刻深度 ....................................................... 105
圖5-12 完成蝕刻之ITO玻璃 ............................................................... 106
圖5-13 完成製作之ITO電阻抗感測電極 ........................................... 106
圖5-14 聚對二甲苯N、C、D材料與化學結構 ..................................... 107
圖5-15 聚對二甲苯沉積過程 .............................................................. 108
圖5-16 聚對二甲苯鍍膜機(PDS2010) ................................................. 109
圖5-17 晶片與晶舟置入聚對二甲苯鍍膜機台中 .............................. 109
圖5-18 反應離子蝕刻機 ...................................................................... 110
圖5-19 完成光阻阻擋層製作之感測電極 .......................................... 111
圖5-20 完成光阻阻擋層製作之金屬接點 .......................................... 111
圖5-21 完成parylene蝕刻之感測電極 ............................................... 111
圖5-22 完成parylene蝕刻之金屬接點 ............................................... 112
圖5-23 金屬接點處之parylene膜厚 .................................................... 112
圖5-24 金屬接點處之ITO電極膜厚 ................................................... 112
圖5-25 ITO電極上方黃金接點膜厚 .................................................... 113
圖5-26 誘導細胞貼附之明膠微圖案製作流程 .................................. 115
圖5-27 完成明膠微圖案製作之感測電極 .......................................... 115
圖5-28 相位差顯微鏡拍攝之感測晶片 .............................................. 116
圖5-29 PDMS矽膠環夾治具設計圖 .................................................... 117
圖5-30 完成加工之PDMS灌注夾治具 .............................................. 118
圖5-31 感測晶片放置於夾治具下圓盤 .............................................. 120
圖5-32 夾治具灌注內壁塗抹PDMS脫模劑 ...................................... 120
圖5-33 電阻抗感測晶片與夾治具組合圖 .......................................... 120
圖5-34 灌注PDMS矽膠於晶片上方 .................................................. 121
圖5-35 真空烤箱烘烤固化PDMS矽膠 .............................................. 121
圖5-36 烘烤後之PDMS矽膠環與感測晶片 ...................................... 121
圖5-37 旋轉拆除夾治具上圓盤 .......................................................... 122
圖5-38 拆除夾治具上圓盤後之感測晶片 .......................................... 122
圖5-39 將PDMS矽膠環由中央金屬環頂出 ...................................... 122
圖5-40 完成PDMS矽膠環製作之電阻抗感測晶片 .......................... 123
圖5-41 PCB電路佈線光罩 ................................................................... 123
圖5-42 PCB訊號連接器 ....................................................................... 124
圖5-43 曝光機進行PCB曝光 .............................................................. 125
圖5-44 PCB顯影定義設計線路 ........................................................... 125
圖5-45 氣泡蝕刻槽 .............................................................................. 126
圖5-46 完成蝕刻之電路板 .................................................................. 126
圖5-47 完成剪裁與開孔加工之電路板 .............................................. 127
圖5-48 電路板與感測晶片結合 .......................................................... 127
圖5-49 完成打線之感測晶片 .............................................................. 128
圖5-50 完成封膠之電路板與感測晶片 .............................................. 128
圖6-1 量測實驗架設示意圖 ................................................................ 130
圖6-2 感測晶片與電路板連接至訊號線 ............................................ 130
圖6-3 自製量測訊號線 ........................................................................ 131
圖6-4 感測晶片放置於光學模組下方 ................................................ 131
圖6-5 充填氯化鈉水溶液之液面 ........................................................ 132
圖6-6 充填含有血清培養液之液面 .................................................... 132
圖6-7 光學模組即時拍攝感測電極 .................................................... 133
圖6-8 平移台之控制盒與平移台控制手把 ........................................ 133
圖6-9 即時拍攝感測晶片上方細胞之畫面 ........................................ 134
圖6-10 產生訊號干擾之掃描結果 ...................................................... 135
圖6-11 培養箱接地後之掃描結果 ...................................................... 136
圖6-12感測電極編號示意圖 ................................................................ 137
圖6-13 工作電極無細胞系統量測 ...................................................... 137
圖6-14 接種八小時之單一細胞貼附於不同工作電極 ...................... 139
圖6-15 電極CH2之單一細胞量測(接種8小時) ................................ 139
圖6-16 電極CH3之單一細胞量測(接種8小時) ............................... 139
圖6-17 電極CH4之單一細胞量測(接種8小時) ............................... 140
圖6-18 HeLa cell接種後24小時(左)與48小時(右) ............................. 141
圖6-19 單一細胞在不同接種時間貼附情形之量測結果比較 .......... 141
圖6-20 貼附面積與阻抗變化量關係圖(細胞接種8小時) .................. 142
圖6-21 兩隻細胞貼附於電極CH10上方(細胞接種8小時) ............... 143
圖6-22 三隻細胞貼附於電極CH12上方(細胞接種8小時) ............... 143
圖6-23 電極CH10之雙細胞量測(細胞接種8小時) ........................... 144
圖6-24 電極CH12之三細胞量測(細胞接種8小時) ........................... 144
圖7-1 無光學補償鏡片所造成之紋路 ................................................ 151
圖7-2 充填不同液體之表面曲面 ........................................................ 152
圖A-1 明膠薄膜厚度、濃度與塗佈轉速之關係 ................................. 164
圖B-1 Z軸電動線性平移台 .................................................................. 165
圖B-2 XY軸電動線性平移台 .............................................................. 166
表目錄
表2-1 明膠薄膜蝕刻率 .............................................. 42
表2-2 光阻薄膜(AZP-4620)蝕刻率 ....................................................... 43
表3-1 不同材料試片之表面接觸角量測 .............................................. 71
表3-2 試片經細胞培養72小時後細胞密度 ......................................... 76
表5-1 實際量測阻值(串聯1 MΩ的限流電阻) ...................................... 97
表5-2 ITO玻璃詳細規格表 .................................................................. 102
表6-1 貼附面積A與阻抗變化量ΔR比較(細胞接種8小時) ............. 142
表6-2 貼附面積A與阻抗變化量ΔR比較(細胞接種8小時) ............. 144
表A-1 明膠薄膜厚度、濃度與塗佈轉速間之關係值 ......................... 164
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