新式細胞培養法：三維明膠鷹架

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姓名

林靜英(Jing-ying Lin) 查詢紙本館藏

畢業系所

生物物理研究所

論文名稱

新式細胞培養法：三維明膠鷹架
(Culturing Cells in Cellular Solids)

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摘要(中)

近年來，科學家在「培養於三維環境的細胞行為表現」方面的研究有快速成長的趨勢。就我們所知，人體內的細胞本生長在由細胞外間質構築而成的三維空間裡，而那些人工製造的三維細胞培養支架，即稱之為「鷹架」。用來產生鷹架的方式有許多，可利用天然的含水凝膠材質（細胞外間質的衍生物）或是鑄模後的孔洞結構來生產，但這些鷹架內的結構組成通常是很不一致。至今，對於何種三維環境是最適合細胞生長的概念也尚未定論。所以在這篇研究中，我們建造了一新式的三維明膠鷹架，它內部的孔洞結構大小一致且排列整齊。方法上我們使用了簡易的微流體裝置，在足夠的氣體流速下製造100μm大小的泡泡。當這些液體泡泡自我排列成結晶狀後，再藉由溫度的變化與化學交聯劑，進行膠化使其成為固體泡沫，以持久性地維持它們的架構。最後以真空打破孔洞間的交界面，即完成內部孔徑大小均一且相通的三維明膠鷹架。另一方面，我們也著手於鷹架內培養細胞，嘗試了三種細胞類型：纖維母細胞，肌母細胞和上皮細胞。不同種類的細胞在鷹架內表現出明顯的形態差異。我們相信這個新式鷹架將能成為一個探討在不同孔徑下細胞行為表現的平台，並有助於針對細胞形態進行更多量化的影像分析。

摘要(英)

In recent years, there is a rapid increase of studies about cell behaviors in three-dimensional (3D) environment. Cells in our bodies are surrounded by 3D environment which is mainly composed of extracellular matrix (ECM). An artificial 3D cell culture support in 3D is often called a scaffold. Various approaches are shown to construct scaffolds such as a natural hydrogel matrix (ECM derivatives) or a precast porous structure whose architecture is often non-homogeneous. There is no consensus on what kind of 3D environment is most suited for cell growth and behaviors. In this thesis, we constructed a gelatin scaffold with a well-defined pore size and crystalline architecture. We used a simple flow-focusing microfluidic device to generate bubbles about 100μm in diameter and high enough air fraction rate. The bubbles self-assembled into crystalline face and we fixed the structure by changing the temperature. The congealed solid foam was further permanently crosslinked by chemical crosslinkers and the facet between pores were ruptured by vacuum. The final scaffold was a monodisperse solid foam with open cells. Furthermore, we cultured fibroblasts, epithelial cells and myoblasts in our 3D scaffolds. Different cells showed distinct morphologies in the scaffolds. We believed that this scaffold could provide a platform to study cell behaviors at different pore sizes and allow more quantitative image analysis for cell morphology.

關鍵字(中)

★ 細胞形態
★ 細胞培養
★ 三維明膠鷹架
★ 微流體裝置

關鍵字(英)

★ microfluidic device
★ cell morphology
★ cell culture
★ gelatin scaffold

論文目次

Introduction 1
The 3D environment in the human body 1
3D cell cultures 2
Experiments 7
Materials and Chemicals 7
Fabrication of Scaffolds 7
Cell Cultures 10
Cell types 10
Substratum-based 2D and 3D culture 11
Immunofluorescence staining and imaging 12
Results 13
Cell Cultured in the gelatin scaffold 13
NIH/3T3 (fibroblasts) showed spindle-shaped in gelatin scaffolds 13
C2C12 (myoblasts) showed tubular morphology in gelatin scaffolds 16
MDCK (epithelial Cells) exhibited cyst-like pattern in gelatin scaffolds 18
Conclusion 24
Reference 25

參考文獻

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14. Guo, Q.S., et al., The microenvironmental determinants for kidney epithelial cyst morphogenesis. European Journal of Cell Biology, 2008. 87(4): p. 251-266.

指導教授

林耿慧(Keng-hui Lin)

審核日期

2010-7-6

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