組織工程用支架表面之細胞遷移行為研究

Cell migration research in the surface of scaffold for tissue engineering

張恆鐘(Heng-Chung Chang)

指導教授 ： 曾清秀

國立中央大學/工學院/機械工程學系/碩士(2007年)

摘要

組織工程用支架提供細胞貼附生長的環境，支架之材料選擇、外型結構與孔洞大小等優劣評估方式，是以最終細胞組織之生化檢測值為依據，然而細胞在支架表面生長與遷移行為也可視為一種評估方式，因此本研究以細胞生長與遷移過程之影像資訊，使用影像處理方式分析擷取出細胞在支架表面上的位移方向、移動速度等細胞生長訊息，以評估支架材料選擇、結構設計之好壞，也可進ㄧ步地探討生物反應器注流之剪應力對細胞生長的影響。
實驗中支架材料使用組織工程中常用的生醫材料:聚乳酸-甘醇酸(Poly-Lactic-Co-Glycolic Acid, PLGA)；細胞之選擇為老鼠軟骨細胞株(Immortalized Rat Chondrocyte, IRC)。為觀察活細胞在支架上分佈影像，採用基因改質之腺病毒(Adenovirus)感染細胞，使細胞帶有綠螢光蛋白(Green-Fluorometer-Protein, GFP)，進而能在倒立式顯微鏡下觀察。在本研究可分為三部份：1. 靜態培養研究：觀察細胞在支架上生長情況，得到細胞在支架上佈滿的過程。2. 細胞遷移研究：採用靜態培養支架上細胞、動態培養液注流支架表面細胞，以觀察細胞遷移情況，得到細胞在支架上遷移訊息。3. 生物反應器之注流研究：觀察不同流速對細胞生長的影響，進而了解細胞與剪應力間的關係，並找出細胞所能承受最大剪應力為何。結果發現細胞在78 μm/sec流速下會受剪應力影響而改變貼附型態，而56.75 μm/sec流速為細胞改變型態之臨界，其剪應力值為0.89 mPa，在28 μm/sec流速時細胞受流場影響平行排列。

關鍵字

剪應力 ； 注流式培養 ； 組織工程支架 ； 綠螢光蛋白 ； 細胞遷移

並列摘要

The tissue engineering of scaffold provided cell attachment conditions. Present to decide the scaffold materials, structure, and pore size methods by final biochemical examined values. Nevertheless, cell migration and proliferation process on scaffold surface is another one of method to decide scaffold design. We researching in cell migration process of information, using image processing method to find cell migration direction and move speed. This information also estimated that the bioreactor’s perfusion shear stress effect cell proliferation.
The experiment of scaffold use poly-lactic-glycolic acid (PLGA). It is common biomedical material using on tissue engineering. The immortalized rat chondrocyte (IRC) chooses as experiment cells. To observed the cells distribution on scaffold surface. Using transgenic of adenovirus infect cells that had Green-Fluorometer-Protein. The attachment of cells on scaffold surface would observe at Inversion Fluorescence Microscope. This research can be divided into three parts: 1. Static cell culture methods: Observe cell grow situation at support, it get cell''s course of covering with the scaffold. 2. Cell migration of research: Using static cell culture of scaffold''s surface cells or the perfusion of medium on scaffold plane, observed the cell migration situation. It will get cell migration direction and move speed. 3. The bioreactor’s perfusion research: Observed the changes of flow speed effect cell proliferation, and then understands the relation among cells and shearing stress, in addition finding out cells can bear the greatest shearing stress. Our findings showed that the cell had change morphology and attachment at a fluid velocity of 78 μm/sec. The cell had original morphology critical limit fluid velocity is 56.75 μm/sec. The shear stress value in the situation is 0.89 mPa. At a fluid velocity of 28 μm/sec, the cell arranged parallel with the flow direction.

並列關鍵字

Cell migration ； Tissue engineering scaffold ； Green-Fluorometer-Protein ； Shear stress ； Perfusion culture

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