細胞在注流式生物反應器之生長研究

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劉貴助(Kuei-Chu Liu) 查詢紙本館藏

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機械工程學系

論文名稱

細胞在注流式生物反應器之生長研究

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★ 週期式圓錐平板裝置之流場分析	★ 設計與製作圓錐平板型生物反應器

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摘要(中)

組織工程是嘗試在活體外培養出組織或是器官，再移植到活體內以解決異體移植時的排斥現象，並且縮短等待捐贈者器官或組織的時間，減小在等待時間中死亡的風險。
早期學者使用靜態單層培養細胞，發現細胞的生長與型態並不好，且細胞無法堆疊成三維的構造，所以使用生物相容性材料製成的三維支架，讓細胞可以堆疊成立體構造，但是發生養份和氧氣傳輸受到限制，使得支架內的細胞無法生長，導致細胞不均勻的分佈，許多學者發展出各種不同形式的動態生物反應器，取代原本使用靜態培養的方式來培養細胞，將養份和氧氣以流體動力學的方式傳輸到支架內部，使細胞分佈較為均勻。
本文利用注流式生物反應器來培養軟骨細胞，設定3種不同流速(11 μm/s、76 μm/s、252 μm/s)培養軟骨細胞以及變換培養液流動方向，可以使細胞在三維的支架中均勻的生長，並可以讓細胞數快速的增加，其中3天培養以培養液雙向流動流速252 μm/s的培養出細胞數最多和細胞分佈最好，9天培養液單向流動培養也是以流速252 μm/s的培養結果最佳，這將有助於縮短生成軟骨成骨的時間。

摘要(英)

Tissue engineers have been trying to grow tissue implants in vitro. Because cells grown in monolayer culture cannot form three-dimensional tissues, and the cells in culture may dedifferentiate, becoming different in phenotypes from native cells, researchers have designed three- dimensional porous scaffolds, on which cells can be seeded, adhere and proliferate into three-dimensional tissues.
Nutrient delivery to cells throughout the cell-seeded scaffolds grown in static culture must occurs by diffusion. As a result, cells on the scaffold periphery are typically viable while cells within the constructs may be less active or even necrotic. To overcome this disadvantage, tissue culture systems that use fluid flow to improve nutrient transports are designed to enhance the cell growth in vitro.
This thesis is to design a perfusion bioreactor for chondrocyte culture in vitro. Culture media are forced to flow by a roller pump through the culture chambers, within which cell-scaffold constructs are placed. Cells are grown with unidirectional and reversal flow respectively at three different perfusion rates (11 μm/s, 76 μm/s and 252 μm/s). Results show perfusion with reversal flow at the veloctiy 252μm/s has the best result for three days of culture. In nine days of culture, unidirectional medium flow at 252 μm/s is better than the other two culture perfusion rates. This study reveals that perfusion can enhance cellular proliferation and improve cartilage forming with carefully controlled perfusion rates.

關鍵字(中)

★ 生物反應器

關鍵字(英)

★ perfusion bioreactor

論文目次

中文摘要
英文摘要
誌謝
目錄 ………………………………………………………………..Ⅰ
圖目錄 ……………………………………………………………..Ⅳ
第一章緒論 ………………………………………………………..1
1.1 文獻回顧 …………………………………………………...3
1.2 研究動機 …………………………………………………….6
第二章生物反應器之設計 ………………………………………..8
2.1 設計概念 …………………………………………………….8
2.2 培養室設計 ………………………………………………..10
2.2.1 流場分析 ……………………………………………...10
2.2.2 流速設定方法 ………………………………………….11
2.3 流體通過支架之壓力差測量………………………….....13
2.4 支架滲透度測量 …………………………………………..14
第三章實驗方法 ………………………………………………….22
3.1 支架材料與尺寸 …………………………………………..22
3.2 細胞來源 …………………………………………………..22
3.3 支架消毒與細胞種植 ……………………………………..23
3.4 利用生物反應器培養 ……………………………………..24
3.5 結果分析 …………………………………………………..25
3.5.1 以DNA含量推估細胞數 ……………………………....25
3.5.2 GFP病毒染色 …………………………………………..27
3.5.3 拍攝LV-SEM …………………………………………….28
3.5.4 統計分析 ……………………………………………….39
第四章結果與討論 ……………………………………………….34
4.1 培養3天結果 ……………………………………………...34
4.1.1 靜態培養 ……………………………………………….34
4.1.2 動態培養 ……………………………………………….36
4.2 培養9天結果 ……………………………………………...39
4.2.1 靜態培養 ……………………………………………….40
4.2.2 動態培養 ……………………………………………….40
第五章結論 ...............…………………………………..58
參考文獻 …………………………………………………………..61
附錄A 實驗藥品及儀器…………………………………………...65
附錄B 細胞數與DNA關係式…………………………………......68
附錄C 統計方法…………………………………………………...69

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指導教授

鍾志昂(Chih-Ang Chung)

審核日期

2006-11-23

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