微流體技術應用於心血管組織工程及細胞遷徙觀察

The applications of microfluidics in cardiovascular tissue engineering and cell migration observation

梅振群(Jeng-Chun Mei)

指導教授 ： 游佳欣

國立臺灣大學/工學院/化學工程學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10161

摘要

微流體技術應用於化工與生化研究領域，佔有舉足輕重地位。另一方面，了解細胞行為的重要原則是能在in vitro條件下模擬in vivo環境培養細胞。因此，相較於二維環境(例如:培養皿)，微米等級的細胞更適合培養在三維的微環境中。由於微流體通道與細胞具有相同尺度(微米級)，本研究中，我們利用微流體技術製造細胞培養的三維鷹架。在三維鷹架中培養心肌細胞與血管內皮細胞，以期修復受損的心血管組織；在其中培養魚鱗細胞，了解細胞在三維鷹架中的移動遷徙現象。
第一部分，初代心肌細胞與內皮細胞培養於由明膠與膠原蛋白製成的三維鷹架。首先，利用微流體通道製造微米泡泡，藉微泡堆疊，形成明膠與明膠-膠原蛋白兩種不同的三維細胞培養鷹架。除測試兩種鷹架的機械性質外，利用活細胞影像技術，發現心肌細胞在三維鷹架相對傳統培養皿能保有更長跳動期間。免疫染色結果也證明，心肌細胞能在三維明膠鷹架維持in vivo型態。根據共軛聚焦顯微鏡結果，發現血管內皮細胞圓形排列於兩種鷹架中。綜合來說，此三維細胞培養平台對於心臟修復與血管新生具有很大應用潛力。
第二部分則是將初代魚鱗細胞培養在三維環境追蹤細胞遷徙軌跡。同樣地，利用微流體技術產生明膠微泡，並將微泡單層排列於另一微流體通道，加入細胞後，在光學顯微鏡下觀察細胞遷徙。細胞遷徙軌跡與型態變化利用活細胞影像技術記錄觀察。此外，分別觀察與比較在二維、三維環境中，魚鱗細胞在電場作用下的趨電性、遷徙速率及速度。了解魚鱗細胞在電場作用下，二維、三維環境中的遷移現象，能提供更多有利資訊，應用於傷口修復等細胞遷徙相關機制，進一步認識in vivo的電生理環境。

關鍵字

三維細胞培養 ； 心肌細胞 ； 心血管組織工程 ； 細胞遷徙 ； 微流體

並列摘要

Microfluidics plays a critical role in many chemical engineering and biochemistry applications. On the other hand, to understand cell behaviors, the important principle of in vitro cell culture is to mimic the in vivo cell grown environment. Therefore, the micro-sized cells are more suitably cultured in three dimensional microenvironment than two dimensional microenvironment ( ex : culture dish ). In this work, 3D scaffold for cell culture was built by microfluidics, due to the comparable size. Cardiomyocytes and bovine aortic endothelial cells ( BAEC ) were cultured for repair of cardiovascular tissue, and keratocytes were seeded for observation of cell migration.
In the first part, primary culture cardiomyocytes and BAEC were cultured in 3D multilayer scaffold, made of gelatin and collagen. At first, gelatin and gelatin-collagen scaffold were produced by formation of microbubbles through the microfluidic device. The mechanical properties of gelatin scaffold and gelatin-collagen scaffold were measured. By live cell imaging, we found that 3D scaffold could prolong the contraction behavior of cardiomyocytes compared with conventional 2D culture dish. Furthermore, the fluorescence staining results assured that cardiomyocytes could maintain in vivo morphology in 3D gelatin scaffold. According to the confocal images, endothelial cells formed circle in both 3D gelatin and gelatin-collagen scaffold. To sum up, this 3D platform for cell culture has promising potentials for heart repair and angiogenesis.
In the second part, keratocytes were harvested through primary culture from Hpsophrys nicaraguensis (a fish). To construct 3D culture system for tracking cell migration, microfluidic techniques were applied to generate microbubbles composed of gelatin, and then the bubbles were fabricated into monolayer in the micro-channel which is cell-traceable under phase microscope. Live cell imaging was used to record the migration trajectory and the morphology change. Moreover, galvanotaxis, the tendency of cells directed by an electrical field was studied. The speed and velocity of keratocytes for both 2D and 3D environment were also compared under the condition with electric field. We believe that the understanding of the keratocytes’ migration behavior in 2D and 3D condition under electric field will provide valuable information in succeeding the model system of wound repairs and electrophysiological environment in vivo.

並列關鍵字

3D cell culture ； cardiomyocyte ； cardiovascular tissue engineering ； cell migration ； microfluidics

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