有機光電介電泳晶片及其體外仿肝小葉人工肝組織排列之應用

Abstract

微全分析系統的發展在近二十年來有很長足的發展，因為微機電系統技術(MEMS)的發展，使得對於微米尺度(at the micro- to nano-scale)的粒子操控或生物應用有了新的工具。在微流體實驗室晶片(microfluidic lab-on-a-chip)的發展中，使用電動(electrokinetics)方式也已經成為在微米/奈米裝置中操控分子和流體重要的技術。 我們的研究團隊在過去幾年以體外重建肝臟組織做為研究方向，開發了以介電泳技術以botton up的方式來將細胞排列成二維大面積組織，以黃光微影的技術在玻璃表面上製作設計好的金屬鈦電極，並使用外加交流電在晶片上產生不均勻電場而將細胞排列在電極之上，為了使經過排列的細胞在晶片上可以長久生存，我們同時也整合微流管道系統來提供細胞生存所必須的新鮮培養液，此外，為了改進操控微粒子的便利性，我們也發展了以有機光導材質TiOPc來製作光電鑷夾，並在此技術基礎之上，建立以光圖形來操控粒子及流體的平台。 在本篇研究裡，我們分別討論了體外組織排列及光電鑷夾技術，而以組織工程作為研究的主軸。前者討論了新型肝臟晶片的設計與製作，如何利用尿素和白蛋白檢測技術來展示人工肝組織的代謝功能展現，此二種物質的分泌量在有排列細胞和晶片上成長都有提升，同時應用於快速藥物篩選，對於由kp引起的發炎反應可提高治療效率。後者則討論有機光導材質的光電鑷夾之製作方法，系統架設和操控原理等等，並以之用來操控單一的微米塑膠粒子，測量對單一細胞的光電操控作用力約為4pN，與其他使用a-Si為基材的光電鑷夾系統有相同的單細胞操控能力。懸浮在矽油中的微小氣泡，對氣泡的最大操控力可達150pN，同時也可在十秒內快速大面積的肝細胞排列成組織，朝向體外光電排列人工組織的研究主軸前進。最後本論文也討論了在肝臟晶片上可再努力的方向，和此種光電操控平台的應用限制及未來展望，也說明了該技術在未來發展上的潛力。

The rapid development of micro electro mechanical systems has provided investigators with novel tools with which to investigate microparticle manipulation, and its biological application, at a micro- to nano- scale level. This expands on the development of the micro total analysis system (µTAS) over the previous two decades. In the field of microfluidics and lab-on-a-chip, electrokinetics technology has provided significant methods for microparticle manipulation and flow control. With the aim of reestablishing liver tissue in vitro, the present study developed dielectrophoresis technology with which to pattern cells as two-dimensional artificial tissues over a large area. A titanium electrode pattern was fabricated on the surface of a glass substrate using a photolithography process, which generated a nonuniform electric field; arranging cells in a specific location when suitable external AC voltage and frequency was applied. The integration of a fresh cell culture medium circulation system on this device enabled the maintenance of the long-term viability of cells. This study also used an organic photoconductive material, titanium oxide phthalocyanine (TiOPc), to develop TiOPc-based optoelectronic tweezers for convenient microparticle manipulation and control of flow and particles on this optoelectronic platform. This study has two main focuses: the liver lab chip and optoelectronic tweezers technology, in relation to tissue engineering. This includes the description of new design and fabrication methods of the liver lab chip as well as its functions, as demonstrated by urea and albumin assay. The concentrations of urea and albumin increase in patterned HepG2 cells and chip surroundings. This study further developed a chip-integrated liver pattern for rapid drug screening. This study’s second focus is organic-based optoelectronic tweezers (OET), including the chip fabrication process, system setup, the manipulation/control principle for single polystyrene beads, single cells, and gas bubbles suspended in silicon oil, and cell patterning. The OET force acting on single cells is approximately 4 pN, which is similar to the acting force of the amorphous silicon (a-Si)-based OET device. For manipulating gas bubbles, the maximum OET force is approximately 150 pN. Using the optoelectronic approach, a large area of liver cell patterning can be established within 10 s. The final section of this article describes the future research direction of the liver lab chip and the applications of the optoelectronic platform. The potential of TiOPc-based optoelectronic tweezers is elucidated.