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系統識別號 U0026-3107201323390200
論文名稱(中文) 細胞電特性監測系統之研究
論文名稱(英文) The Study of Cellular Electrical Property Monitoring System
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 101
學期 2
出版年 102
研究生(中文) 盧宜裕
研究生(英文) Yi-Yu Lu
電子信箱 yiyulu@cc.feu.edu.tw
學號 p88931042
學位類別 博士
語文別 英文
論文頁數 104頁
口試委員 指導教授-鄭國順
召集委員-林灶生
口試委員-施東河
口試委員-陳家進
口試委員-周楠華
口試委員-王明習
口試委員-許永和
中文關鍵字 電極陣列  生物電阻抗頻譜技術  細胞數量  電極培養皿  Cole-Cole 圖 
英文關鍵字 Electrode array  Electrical bioimpedance spectroscopy  Cell number  Electrode-based culture  Cole-Cole plot 
學科別分類
中文摘要 在生物醫學的領域中,論述培養中細胞的特性是重要的議題。本研究用於特性化細胞的成長,並使用微機電系統(Micro-Electro-Mechanical-System, MEMS)的技術,首先發展一多電極培養皿監測系統,以三十二個電極,貼附在直徑為15 mm、高為10 mm的培養皿內緣,每個電極寬為1.3 mm,且相鄰電極的間隙為6 mil。其次,使用Cole-Cole圖並結合有著常數相角 (constant phase element, CPE) 的三元件電路模型分析方法,擷取特徵用以分析;並繪出時域的Cole-Cole圖,在26小時後,隨著細胞膜逐漸失去完整性以決定參數如何變化。在26到30小時之間,其最大相角(maximum phase)減少了5%,下降角(depression angle)增加了25%。在細胞衰退時,其他參數的曲線斜率增加,同時R2參數增加為142%。於細胞衰退的研究中,Cole-Cloe圖中的直徑及細胞電路模型中的R2,證明是特別的重要。最後,在PC12細胞成長的研究中,於正常濃度的培養基裡,阻抗與細胞數量之間的相關係數,塗有poly-D-lysine (PDL)的係數為0.868,而沒有塗佈PDL的係數為0.836。因此,阻抗可能做為細胞成長特性的一個指標。所提出的系統,可能提供一種細胞成長與細胞性質改變的監測方法。
英文摘要 The characterization of cell growth during culturing is an important issue in many fields related to biomedicine. In this study, a novel multi-electrode culture monitoring system is firstly developed using Micro-Electro-Mechanical-System (MEMS) technology for characterizing cell growth. There are 32 electrodes attached inside a culture dish 15 mm in diameter and 10 mm in height with the width of 1.3 mm and the gap of 6 mil. Secondly, the Cole-Cole plot method is employed to extract features for analysis using an equivalent three-element circuit model with a constant phase element (CPE). A time-frequency based Cole-Cole plot is used to determine how the parameters changes with the cell membranes gradually lost integrity over a period of 26 hours. The maximal phase decreases an additional 5% and the depression angle increases an additional 25% between 26 and 30 hours. The curve slopes of other parameters increased simultaneously with cell degeneration and R2 values in the cell culture increased to 142%. The diameter used in the Cole-Cole plot and the R2 values in the cell circuit model is proved to be particularly important in the investigation of cell degeneration. Thirdly in the study of PC12 cell growth, the correlation coefficients between impedance and the number of cells were 0.868 (coated with PDL) and 0.836 (without PDL) for a medium of normal concentration. Thus, impedance may be used as an index for the characterization of cell growth. The proposed system may provide a new approach to the monitoring of cell growth and cell property changes.
論文目次 ABSTRACT I
中文摘要 II
誌謝 III
TABLE OF CONTENTS IV
LIST OF TABLES VI
LIST OF FIGURES VII
CHAPTER 1 INTRODUCTION TO THE ELECTRICAL PROPERTIES OF CELLS 1
1.1 BACKGROUND 1
1.2 ELECTRICAL CONDUCTANCE OF LIVING CELL 2
1.3 CELL MONITORING 3
1.4 STUDY FRAMEWORK 4
1.5 MOTIVATION AND PURPOSES 5
CHAPTER 2 LITERATURE REVIEW 7
2.1 THEORY OF ELECTRICAL IMPEDANCE SPECTROSCOPY 7
2.1.1 Bioimpedance representation 9
2.1.2 Bode diagrams 9
2.1.3 Cole- Cole plot 10
2.1.4 Three-dimensional representation 13
2.2 IMPEDANCE SPECTROSCOPY METHODS 15
2.2.1 Electrical properties of biological tissues 15
2.2.2 Equivalent circuit analysis 16
2.2.3 Parameters for Cole-Cole plots 18
2.3 BIOIMPEDANCE IN MEDICAL APPLICATIONS 21
2.3.1Cell behavior reflected in measured impedance 22
2.3.2Culture monitoring systems 22
2.3.3 Multi-electrode impedance sensors 23
2.4 ELECTRICAL PROPERTIES IN CELL CULTURES 23
2.5 BIOIMPEDANCE SPECTROSCOPY IN CELL GROWTH 25
CHAPTER 3 MATERIALS AND METHODS 28
3.1 MONITORING SYSTEM 28
3.1.1 Measurement system for PC12 cell degeneration 28
3.1.2 Measurement system for PC12 cell growth 29
3.2 CELL CULTURE DESIGN OF AN ELECTRICAL BIOIMPEDANCE SYSTEM 31
3.2.1 Fabrication of cell culture electrode array 33
3.2.2 Electrode device assembly 35
3.2.3 Electrodes array testing 36
3.2.4 Summary 42
3.3 SCANNING SYSTEM 44
3.4 INCUBATION SYSTEM 46
3.5 BIOIMPEDANCE ANALYSIS 47
3.5.1 Simulation of electrical impedance spectra 48
3.6 THREE-DIMENSIONAL REPRESENTATION FOR PC12 CELL GROWTH CHARACTERIZATION 52
3.6.1 Three-dimensional perspective plotting 53
3.6.2 An Example of three-dimensional perspective plotting 54
3.6.3 Summary 60
CHAPTER 4 EXPERIMENTAL DESIGN 62
4.1 ELECTRICAL PROPERTY OF PC12 CELLS DEGENERATION 62
4.1.1. PC12 cell line preparation 62
4.1.2. Cell culture with electrode-array 62
4.1.3. Integrated incubation system 63
4.1.4. The EIS system 64
4.1.5. Cell circuit model 65
4.2 CELL GROWTH CHARACTERIZATION USING MULTI-ELECTRODE BIOIMPEDANCE SPECTROSCOPY 67
4.2.1. Multi-electrode culture monitoring system 67
4.2.2. Experimental design 69
4.2.3. Data collection and processing 71
CHAPTER 5 RESULTS AND DISCUSSION 72
5.1 ELECTRICAL PROPERTY OF PC12 CELLS DEGENERATION 72
5.1.1. Simulation of electrical impedance spectra 72
5.1.2. Cell degeneration 74
5.1.3. EIS measurements 75
5.1.4. Cole-Cole plot and parameters 76
5.2 CELL GROWTH CHARACTERIZATION USING MULTI-ELECTRODE BIOIMPEDANCE SPECTROSCOPY 80
5.2.1. EBIS measurement for PC12 proliferation 80
5.2.2. Cell count associated with microscopic images 85
5.2.3. The relation between the bioimpedance and cell proliferation 87
CHAPTER 6 CONCLUSIONS AND PROSPECTS 90
REFERENCES 92
PERSONAL PROFILE 100
LIST OF PUBLICATIONS 101
(A) JOURNALS 101
(B) CONFERENCES 101
(C) PATENTS 103
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