電控微流體平台跨尺度操控顆粒與水膠並建構體外肝臟小葉組織

Abstract

微流道系統、3D列印等微流體技術，發展至今已經相當成熟並被應用於工程、材料以及生物醫學等領域。然而，提及同時操控液體與固體粒子的能力，使用現有的系統與技術仍無法實際達成。在本研究中，我們首先運用電控微流體(Electromicrofluidic platform, EMF)平台以介電濕潤(Electrowetting on dielectric, EWOD)與液體介電泳(Liquid dielectrophoresis, LDEP)驅動不同種類水膠預聚物，並以粒子介電泳(Dielectrophoresis, DEP)排列水膠預聚物中的聚苯乙烯粒子，建構圖案化的微結構，成功展現電控微流平台跨尺度操控的能力。接著，我們將此平台與技術應用於生物領域，依據不同的細胞類型，給予相對應成長微環境，同時藉由電控微流體平台整合不同交聯策略的生物相容性水膠，建構以水膠為基底的仿生細胞培養支架，進行多種細胞的共培養後形成具有適當的細胞表型與功能的體外肝臟小葉組織。而本研究所使用的水膠材料分別為光交聯的Gelatin methacryloyl (GelMA)與Polyethylene glycol diacrylate (PEGDA)、化學交聯的纖維蛋白凝膠(Fibrin gel)以及熱交聯的基底膜基質(Matrigel)。細胞株分別為人類肝癌細胞(Human hepatocellular carcinoma, Huh-7)、人類臍帶靜脈內皮細胞(Human umbilical vein endothelial cells, HUVEC)以及人類單核球細胞(Human monocytic cells, THP-1)。而在仿生組織的培養過程中，我們將觀察細胞的生長以及細胞與細胞之間的交互作用，最後施加會導致肝毒性產生的藥物 (Acetaminophen, APAP)，並量測肝臟細胞白蛋白的分泌改變量與細胞的活性變化，成功地將電控微流體排台應用於體外仿生組織的建構。此外，這個體外仿生肝臟組織可以作為基礎醫學研究、不同種類藥物測試甚至是病原體研究的理想體外模型。在未來，電控微流體平台同時跨尺度操控液體與固體的能力將能夠被應用於其他研究甚至是生醫、材料工程等領域，具極大的開發條件與潛力。

In this study, electromicrofluidic (EMF) platform integrating electrowetting-on-dielectric (EWOD) and dielectrophoresis (DEP) forces is used to pattern multiple hydrogel prepolymers containing solid particles into in-vivo-like hepatic lobule arrangements. Biocompatible hydrogel with different concentration and stiffness was manipulated by the electromicrofluidic platform to construct a hydrogel-based scaffold for cell culture and form an in vitro liver tissue with proper cell morphology and phenotypic function, and our goal is to engineer a liver lobule tissue with blood vessel formation. Therefore, four kinds of hydrogels were used in this research, including photo-crosslinkable hydrogel, gelatin methacryloyl (GelMA) and polyethylene glycol diacrylate (PEGDA), chemically-crosslinkable hydrogel, fibrin gel, and thermally-crosslinkable hydrogel, matrigel. On the other hand, human hepatocellular carcinoma (Huh-7), human umbilical vein endothelial cells (HUVEC) and differentiated human monocytic cells, THP-1 were cultured in biocompatible hydrogels to form the in-vitro biomimetic liver tissue. To construct in vitro hepatic-lobule-like microstructures, Huh-7 and HUVEC mixed with THP-1 were dispersed in GelMA prepolymers respectively with appropriate concentrations. Furthermore, we measured the albumin secretion from different culture conditions and evaluated the function of our liver lobule tissue. For the applications, we investigated the influence of drug hepatotoxicity on liver function. At last, to improve the cell proliferation of HUVEC, we have cultured HUVEC in different extracellular matrixes, expecting that the microvascular formation of endothelial cells may improve the function of our liver tissue. In the future, this engineered tissue can serve as an ideal in vitro model for basic medical research, drug screening and pathogen interaction.