Abstract

In this thesis, an extracorporeal liver tissue vitalizing system is reported. To investigate and test metabolism and accumulation of toxic chemicals in liver tissues, long-term in-vitro culture is very much needed. Presently tissue slices can only receive nutrients on the surface, and tissue degradation is resulted because the core is deficient of nutrients. Liver tissues cannot survive over 3 to 5 days by using conventional static culture method. Conventional static culture method without fresh culture medium supplement does not provide an appropriate environment for culturing liver tissues in-vitro. Therefore, a brand new system must be developed. The extracorporeal liver tissue vitalizing system with a bioreactor is demonstrated. Well-design nozzle-shaped wells in bioreactor are employed. These nozzle-shaped structures bring out extremely high efficiency of nutrient supplements. Liver tissues express better architecture, morphology, and viability by using this on-chip hydrodynamic culture system. A 9-day culture experiment is demonstrated in this research, and there is a great result of longer culture period which can provide more time for doctors to develop cure strategies for each individual patient. For preliminary research, liver tissues of transgenic mice are used in this research. Livers of these transgenic mice secrete HBsAg antigen. By examining the expression of HBsAg antigen, basic liver function remains during culture period. In addition, a Polydimethylsiloxane (PDMS) layer which is cultured with mesothelial cells is employed. Mesothelial cells have been presumed that can secrete some special proteins which are good for liver tissues. To study pathology of liver tissues, we culture liver tissues by using the extracorporeal liver tissue vitalizing system. This hydrodynamic system contains a syringe pump, silicone tubes, and the bioreactor. The syringe pump is set at the optimized flow velocity which can provide enough perfusion pressure without generate destructive shear stress on liver tissue and mesothelial cells. In this research, we develop a system which can prolong lifetime of liver tissues in-vitro. Therefore, patients who are suffered from liver diseases could have an opportunity to find treatments in a short time.

Key words: liver tissue; in-vitro culture; transgenic; nozzle; hydrodynamic
摘要
近年來，生醫工程技術在臨床醫學中扮演重要的角色，目的為突破既有醫學診斷、檢驗上的限制，因此，許許多多如生醫晶片(Biochip)、生物反應器(Bioreactor)等微型化技術相繼誕生，大幅提升了醫療服務的效率與準確性。然而，在肝臟醫學領域，傳統組織體外培養法已達極限，諸如肝臟病理、新陳代謝、B型肝炎、C型肝炎等研究，須仰賴更先進的技術以延長肝臟組織在體外的生存時間。因此，本文提出一體外肝臟組織維生系統，目的為提高肝臟組織於體外長時間培養的生存率，進而達到延長肝臟組織在體外的生存時間，醫生們得以獲得更多寶貴的時間針對不同病人來研擬治療的策略與方針。此系統包含一具有特殊噴嘴型幾何結構的生物反應器，具有高效率將養分帶入肝臟組織，並將代謝廢物攜出的特色。另外，將人類間皮細胞(Mesothelial Cell)培養於一特殊高分子材料聚二甲基矽氧烷(PDMS)，肝臟組織與間皮細胞直接接觸，兩者的交互作用將有利於肝臟組織於體外的培養。本實驗使用基因轉殖鼠(Transgenic Mice)之活體肝臟組織，該組織能生產B型肝炎表面抗原，文中描述肝臟組織於九天期體外培養實驗的過程與檢驗結果，檢驗其B型肝炎表面抗原的表現量、組織化學染色切片(H&E Stain)以及細胞凋亡檢測(TUNEL Assay)。結果顯示，透過此生物反應器與流體動力學搭配之體外肝臟組織維生系統，肝臟組織表現出較高生存率。然而，使用傳統靜態培養法的組織，在組織結構、細胞形態以及最重要之生存率方面，表現均不如使用體外肝臟組織維生系統。全世界每年有極多的B型肝炎、C型肝炎病患，每一位病患的病徵也不盡相同，如何針對單一病患提供適切的治療方式，是未來重要的目標之一。因此，體外肝臟組織維生系統提供一獨立的操作平台，肝臟組織在此平台下的存活時間大幅超越當前傳統的組織培養方法，為未來藥物測試及病理相關研究創造出更多的可能性。



關鍵字: 肝臟組織; 體外培養; 基因轉殖; 噴嘴;流體動力學

Table of Content
Chapter 1 Introduction 1
1. 1 Background of Tissue Engineering 1
1. 2 Category of Hepatocyte Assay System 8
1.2.1 Monolayer Hepatocyte 8
1.2.2 Suspended Hepatocyte 9
1.2.3 Tissue Slices 10
1. 3 Microfluidics 18
Chapter 2 ELViS System Development 20
2.1 Extracorporeal Liver Tissue Vitalizing System(ELViS) 27
2.2 Chip Design and Working Principle 30
2.2.1 Single unit 34
2.2.2 4×3 Array 35
2.3 Simulation Analysis 37
2.3.1 Array Simulation 37
2.3.2 Nozzle Angle Simulation 38
Chapter 3 Fabrication 40
3.1 Machining Process and Fabrication Results 40
Chapter 4 Experiments and Results 42
4.1 ELViS System Setup 42
4.2 ELViS Perfusion Test 44
4.3 Experimrntal Results 45
4.3.1 HBsAg Expression 46
4.3.2 H&E Stain 49
4.3.3 TUNEL Assay 53
4.4 Discussion 68
Chapter 5 Conclusions 72
5.1. Summary 72
5.2. Applications 74
References 75

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