Development of a Microfluidic Chip for Construction of Hepatic-Lobule Mimetic Tissues

仿肝小葉結構之體外組織培養微流體生物晶片開發

秦重光(Chung-Kuang Chin)

指導教授 ： 劉承賢

國立清華大學/工學院/動力機械工程學系/碩士(2010年)

https://doi.org/10.6843/NTHU.2010.00053

摘要

In recent years, liver failure to cause death is always a critical issue. Particularly in Taiwan, the incidence of liver diseases is higher even more and has been ranked on top in the world. Actually, about the field of (hepatic) tissue engineering, owing to the shortage of liver organ source in surgical reconstruction medicine and the safety problem about infection and autoimmunity, the emphasis of this field has been gradually shifting from the conventional clinical therapies-organ transplants to the way we generate engineered tissue for study of human tissue physiology and pathophysiology in-vitro. Therefore, develop an in-vitro engineered liver tissue here to serve as an excellent model system for the study of liver-related issue, like liver regeneration, ischemia/reperfusion injury, fibrosis, microbial infection, and inflammation, is significant for prolonging patient's lives.
This study will present the design, microfabrication, and implementation of our dielectrophoretic (DEP)-based cell-patterning microfluidic platform, which includes triplicate analysis microfluidic channel design for approaching the requirements of experiment consistency and the biological statistics analysis in our chip, and the microchamber with microelectrodes for in-vitro engineered lobule-mimetic liver tissue reconstruction. The combination of MEMS technology and well-developed DEP-based cell-patterning enable the regeneration of complicated liver tissue to be easier and reproducible. In addition, to evaluate how the stress of DEP manipulation on cells, which is a key issue about this method, we performed FDA/EtBr assay to assess the cell viability and acquired the survival percentage of cell, after DEP operation, is 95 %. Moreover, from the drug test for metabolism studies, we also demonstrated the enhancement of hepatocyte activity, 30 % upgrading, by our engineering arrangement of lobule-mimetic liver tissue, which supports our biomimetic engineering concept about mimicking the cell-cell interactions and genetic architecture for preserving cell activity and the specific and complex functions of complicated tissue.
We anticipate this liver-on-chip engineering to be a starting point and direct for more sophisticated tissue regeneration and a platform for extracting the biology information from engineer tissues in the future.

關鍵字

組織工程 ； 介電泳 ； 細胞排列 ； 微流道 ； 肝小葉 ； 代謝

並列摘要

近年來，肝病致死一直是個棘手的問題，特別在台灣，發生肝臟疾病的比例更是高，位居全球之冠。在肝臟組織工程的領域，過往由於在器官移植的進行上，肝臟來源長期不足和病毒感染、自體免疫排斥等問題，近來的發展重心漸漸朝向於體外的肝臟組織重建和研究，依此模式有助於我們對人體內的行為機制有更進一步的認識，也對於研究肝臟相關的疾病和反應以達到免疫、防治和延長病患的壽命的目標。
本研究，我們提出一新穎的微流體細胞排列晶片，透過微流道的設計，滿足適合生物實驗，多重複及統計分析的需求，同時藉由電極圖形的設計，利用介電泳效應，成功完成仿體內肝小葉(肝臟的基本單元)圖形的工程肝臟組織體外重建。透過整合微機電和介電泳細胞排列技術，我們將複雜的肝臟組織重建變得容易且具再現性。利用介電泳效應做細胞操控，最主要的關鍵在於操作過程中，細胞所受到的傷害程度，這裡我們利用FDA/EtBr試劑對細胞的存活率做評估，結論指出細胞在操控後的存活率依然相當的高，有95 %。另外，透過藥物實驗分析，我們進一步探討了不同條件下細胞的代謝活性，結果證實經過工程操控(仿建肝小葉)後的肝臟細胞，活性提升了30 %，這個結果支持了我們這樣的仿生工程，藉由仿建體內細胞環境來重現組織功能、建構器官的概念。
我們很期待這樣的肝組織晶片技術將來可幫助重現更多、更複雜的組織器官，同時也可用於探討更多生物相關議題的平台。

並列關鍵字

Tissue Engineering ； Dielectrophoresis (DEP) ； Cell patterning ； Microchannel ； Hepatic lobule ； Metabolism

參考文獻

[4] L. G. Griffith, and G. Naughton, “Tissue engineering--current challenges and expanding opportunities,” Science. 295(5557), pp. 1009-14, (2002).

[5] L. G. Griffith, and M.A. Swartz, “Capturing complex 3D tissue physiology in vitro,” Nat Rev Mol Cell Biol. 7(3), pp. 211-24, (2006).

[6] Y. Nahmias, F. Berthiaume, and M.L. Yarmush, “Integration of technologies for hepatic tissue engineering,” Adv Biochem Eng Biotechnol. 103, pp. 309-29, (2007).

[7] C. T. Ho, R. Z. Lin, H. Y. Chang, and C. H. Liu, “In-vitro rapid centimeter-scale reconstruction of lobule-mimetic liver tissue employing dielectrophoresis based cell patterning,” in Digest Tech. Papers Transducers‘07 Conference, vol. 1, pp. 351-354, (2007).

[8] WTEC Panel Report on Tissue Engineering Research, International Technology Research Institute, Jan, (2002).