微流體剪應力對內皮細胞與多能羊水幹細胞分化之影響

Abstract

為增進全人類健康的發展，位居現代人最大死因的心血管疾病與最具發展潛力的幹細胞等研究正如火如荼的在各領域中熱烈進行著。微機電技術發展的微小化、輕量化與可攜化的細胞晶片系統，有助於揭開各項細胞生物資訊與了解心血管致病原由。本研究目的在建立一套結合培養與即時觀測功能的陣列化生物反應晶片系統，並將其應用於血管內皮細胞與多能羊水幹細胞的培養分化研究上。研 究方法可分為微流道晶片本體的設計與製造、建構藥品與流體剪應力供給系統、建構光學即時觀測系統、進行細胞剪應力實驗與多能羊水幹細胞引導分化實驗。 本研究中發現特定流體剪應力值作用一天後，內皮細胞在直線型、45°折線型與三叉型的流場中有按照流線方向進行排列；3、6、12 與18 dynes/cm2 四種流體剪應力於水平直線流場作用下一天後，分析內皮細胞與水平流場間角度的差異，其中發現在12 dynes/cm2 的流體剪應力作用下，在一天後有高達八成以上的內皮細胞與水平流場間角度在20°以內，並統計各流體剪應力值下內皮細胞遷移的速度，發現3 與6 dynes/cm2 作用下遷移速度約為0.4 um/min，12 與18 dynes/cm2 作用下遷移速度約為0.8 um/min；利用內皮細胞生長因子搭配12 dynes/cm2 的流體剪應力引導多能羊水幹細胞12 小時後，多能羊水幹細胞表現出vWF 此種內皮細胞生物標記，並即時的由顯微系統獲得影像證據。 有別於過去大多數對於血管新生研究中所使用的大型機台，本研究將這類實驗由公分尺寸的等級縮小為更類似真實人體血管管徑大小的微米尺寸等級，解決上述大型機台實驗中需要大量培養液或藥品等缺點，並充分利用微流道中良好層流的特性，建立穩定的仿生實驗流場，減少各種實驗變因，驗證了本系統未來應用於心血管疾病研究與幹細胞分化研究上之可行性。

Research of stem cells for cardiovascular diseases is of growing interest. The miniaturized and portable micro bio-devices fabricated by micromachining technology is able to provide in-depth understanding of biological mechanism and activities related to cardiovascular diseases. The objective of this research is to study the influence of microfluidic shear stress to the culture of ECs, and AFSC differentiation to ECs. Design and fabrication of micro devices are key to success for the experiment, as well as the integration of real-time monitoring optical and culturing systems. The experiment of cell culture was carried out over 24 hours. It was found that, under the microfluidic stimulation of shear stress, endothelial cells were successfully adhered on the substrate, stretched and aligned along with constant flows applied. The microfluidic shear stresses were made at 3, 6, 12, 18 dynes/cm2, respectively. This work shows the result of cell morphology and cell arrangement under the fluidic shear stress. 80% of endothelial cells were mostly found in morphology and arrangement aligned along the flow direction. Meanwhile, stretched cells were found in less than 20° out of the flow direction at the fluidic shear stress of 12 ynes/cm2. In addition, the migration speed of endothelial cells was also measured to be about 0.4 um/min. at the shear stress of 3 and 6 dynes/cm2, and 0.8 um/min. at the shear stress of 12 and 18 dynes/cm2, respectively. Biological and fluidic stimulations of AFSC ifferentiation to endothelial cells were also conducted. vWF bio markers were first found in micro devices to provide initial evidence of AFSC differentiation to endothelial cells. At last, the device and system were successfully established and utilized for its potential of AFSC differentiation to endothelial cells for cardiovascular diseases.