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Title: | 電控微流體平台建立體外肝臟小葉微環境作為藥物肝臟毒性分析 In Vitro Hepatic Lobule Microenvironment via Electromicrofluidic Platform for Drug Hepatotoxicity Assay |
Authors: | Jyong-Huei Lee 李炯輝 |
Advisor: | 范士岡(Shih-Kang Fan) |
Keyword: | 電控微流體,體外仿生肝臟小葉組織,水膠硬度,藥物肝臟毒性分析, electromicrofluidics (EMF),hepatic lobule tissue,hydrogel,drug hepatotoxicity assay, |
Publication Year : | 2017 |
Degree: | 碩士 |
Abstract: | 在本研究中,我們運用電控微流體(EMF)平台跨尺度的以介電濕潤與液體介電泳驅動含有不同細胞的水膠預聚物溶液,並以粒子介電泳驅動細胞,排列成體內仿生肝臟小葉組織的型態,而後交聯形成水膠。依據不同的細胞類型,給予相對應物理微環境,建構以水膠為基底的仿生細胞培養支架的異質細胞培養微結構,形成具有適當的細胞形態和表型功能的體外肝組織。現今許多研究團隊運用各式各樣的工程技術,透過體外培養的方法,建構出具有一定功能的仿生體外組織與人體器官晶片。相較於三維的組織,肝臟細胞在二維的培養環境下,會喪失其肝臟細胞原有的型態與功能,因此這些技術需要搭配具有生物相容性的材料,透過模仿細胞外間質的特性,提供細胞仿生的體外培養環境,而三維體外組織的養分與代謝物如何輸送變成一項難題,因此我們希望藉由體外血管增生,運輸細胞所需的養分與代謝物。本研究所使用的水膠材料包括Gelatin methacryloyl (GelMA)與Poly(ethylene glycol) diacrylate (PEGDA)兩種光交聯水膠,並加入熱交聯性質的水膠Matrigel建構三維水膠異質結構。本研究所用到的細胞株分別為人類臍帶靜脈內皮細胞(HUVEC)、人類肺臟纖維母細胞(HFL1)、人類單核球細胞(THP-1)與人類肝癌細胞(HepG2, Huh-7)。首先仿肝臟小葉圖案的電極,以頻率為1 kHz的電壓訊號同時操控兩種不同平均分子量的PEGDA。並且透過具有隙縫的長條狀電極,利用頻率為4 kHz的電壓訊號以介電泳力排列粒子,成功展示了電控微流體平台跨尺度操控的能力。接著量測我們所用來包覆內皮細胞(10% GelMA)與肝臟細胞(5% GelMA)的水膠硬度,確認我們的水膠硬度與文獻相符。並且測試提升肝臟細胞分泌酵素Cytochrome P450的藥,rifampicin (提升CYP 3A4)的效果,探討藥物acetaminophen對於肝臟細胞與內皮細胞的毒性。最後透過仿肝臟小葉圖案的電極,以頻率為1 kHz的電壓訊號操控含有血管內皮細胞與肝臟細胞不同濃度的水膠,給予不同硬度的水膠微環境。並利用頻率為4 kHz的電壓訊號以介電泳力排列HUVEC細胞,建構仿肝臟小葉三維異質微結構,在培養13天之後,利用免疫螢光染色研究圖案化細胞共培養對於細胞型態的影響,期望此仿肝臟三維異質水膠細胞培養微結構成為具有一定水平的生理功能的組織。並在第10天加入藥物rifampicin,提升此肝臟組織CYP 3A4的分泌量。接著在第12天,加入藥物acetaminophen,而這個藥物會與CYP酵素反應,探討藥物與藥物之間的交互作用,分析我們建立的體外仿生肝臟小葉組織,對於藥物毒性的反應,比較二維共培養、三維共培養以三維圖案化的仿生肝臟組織,在加了藥物的白蛋白的分泌量,我們觀察到,沒有圖案化的二維共培養及三維共培養在加入有肝臟毒性的藥一天之後,白蛋白分泌量仍有緩慢上升,而我們所建立的三維仿生肝臟小葉組織的白蛋白分泌量卻是下降的,這個數據顯示我們的三維仿生肝臟小葉組織,對於藥物是有比較高的靈敏度的。在未來,這個體外仿生肝臟組織可以作為基本醫學研究,藥物篩選,病原體研究的理想體外模型。這個平台也是個性化醫療作為藥物測試和開發工具的潛在候選者,以達到再生醫學和人體器官晶片的願景。 Electromicrofluidic (EMF) platform integrating electrowetting-on-dielectric (EWOD) and dielectrophoresis (DEP) forces is used to pattern multiple hydrogel prepolymers containing different cell types into in-vivo-like hepatic lobule arrangements in one step. Recently, biofabrication techniques that improve the physiological functions of cells cultivated in vitro by providing them properly engineered microenvironments imitating their anatomic situation. From several studies, the cells in three dimensional (3D) culture including hepatocytes better maintain their original morphologies and phenotypes comparing with those in 2D culture. In this thesis, 3D biomimetic liver tissues are fabricated with biocompatible hydrogels serving as the extracellular matrices. Endothelial cells are arranged and cultivated in the liver tissues for potential nutrients and metabolites transportation across the 3D tissues. Photo-crosslinkable hydrogels, gelatin methacryloyl (GelMA) and poly (ethylene glycol) diacrylate (PEGDA), and a thermally crosslinkable hydrogel, Matrigel, were successfully manipulated on the EMF platform. Human umbilical vein endothelial cells (HUVEC), human lung fibroblasts (HFL-1), human monocyte cells (THP-1), and human hepatocellular carcinoma cells (HepG2, Huh-7) were used to form the in vitro biomimetic liver tissues. We constructed 3D tissues consisting of multiple hydrogels of appropriate stiffness and with cells by simultaneously controlling hydrogel prepolymer fluids and cells with appropriate electrical signals on the EMF platform. Furthermore, the effect of rifampicin on Cytochrome P450 3A4 and the toxicity of acetaminophen were investigated. Stiffer 10% GelMA prepolymer with hepatocytes was patterned into hepatic lobule, while the HUVEC cells were patterned in the more compliant 5% GelMA prepolymer. The crosslinking was accomplished by UV exposure. Morphology and proliferation of cells were observed inside the 3D tissue by immunofluorescence staining. To enhance the secretion of Cytochrome P450, rifampicin was added after 10 days in coculture. Furthermore, to study the interaction between rifampicin and drugs, acetaminophen was added on day 12. Acetaminophen would react with CYPs and generate hepatotoxicity. On day 13, the albumin secretion of hepatocytes in 2D none patterned coculture and in 3D none patterned coculture kept rising, but the albumin secretion of hepatocytes in our in vitro hepatic lobule tissue declined. We consider that our in vitro hepatic lobule tissue has better sensitivity to the drug toxicity. In the future, the tissue can serve as an ideal in vitro model for basic medical research, drug screening and pathogen interaction. This platform is also a potential candidate for personalized medicine in drug testing and development and even for regenerative medicine and organs-on-a-chip. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77835 |
DOI: | 10.6342/NTU201703973 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 機械工程學系 |
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ntu-106-R04522115-1.pdf Restricted Access | 7.19 MB | Adobe PDF |
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