以微流道進行三維培養人類乳癌細胞及其在抗癌藥物的篩選應用

Three Dimensional Culture of Human Breast Cancer Cells in Microfluidics and Its Application in Screening of Anticancer Drugs

林帝宇(Ti-U Lin)

指導教授 ： 謝明發

中原大學/工學院/生物醫學工程研究所/碩士(2015年)

https://doi.org/10.6840/CYCU.2015.00077

摘要

本研究以微影製程技術製備三維培養人類乳癌細胞株的微流道晶片，具有上下流道的雙層微流道接著於7.5x2.5 平方公分的玻璃基板上，先以SU-8光阻製作公模，再使用PDMS翻模製備凹槽式上下流道。在公模製作中，以旋轉塗佈法製作平均厚度325.75 ± 5.55 μm之SU-8層。為三維培養細胞於微流道中，微流道底部填充玻尿酸與膠原蛋白的細胞外間質材料；本研究合成光交聯型甲基丙烯酸環氧丙酯改質玻尿酸；將甲基丙烯酸環氧丙酯在鹼性條件下接枝到玻尿酸D 型葡萄醣酸醣基的羧酸，以質子核磁共振光譜之圖譜計算，平均接枝率為 21.03 %。豬皮萃取的膠原蛋白以聚丙烯醯胺膠體電泳鑑定測量的相對分子量；在250 kDa有β-chain，在100-130kDa有α-chain，以膠原蛋白含量檢測法測量萃取的膠原蛋白濃度為0.48 mg/mL，換算成純度為95.8%。未充填細胞外間質的微流道以75%酒精和365 奈米的UV光照射3小時後以無菌水灌流液於細菌培養箱37 oC下固態培養48小時可達無菌狀態，將人類乳癌細胞填充於微流道中24小時貼附後以注射針幫浦施打8 μL/min的MEM培養基，細胞存活良好。微流道置放於倒立式顯微鏡載台安裝的二氧化碳細胞培養系統，經過6小時後系統可以維持在37 ± 0.1oC並持續培養120小時以上，以 Hypoxyprobe™-1 Plus kit檢測人類乳癌細胞在5% CO2以及37 oC 條件下並未偵測到缺氧綠螢光蛋白的表現。在二維培養皿培養與三維微流道培養細胞檢測方面，以LDH assay 檢測1至5天發現，其細胞死亡率都不超過35%。使用星狀聚己內酯-聚乙二醇-葉酸共聚合物(4SPCL100-PEG-Folate) 做為藥物載體包覆抗癌藥物-艾黴素，並製備艾黴素微胞(DMC100F)。使用動態光散射分析結果得到微胞粒徑為132.1 奈米。使用紫外光可見光光譜儀分析藥物的包覆率為87.1 %。DMC100F在PH 7.4的中性磷酸鹽緩衝液中經過60小時釋放率為21.3 %，在PH 4.9 的酸性磷酸鹽緩衝液狀態下釋放78.6%。從半致死劑量(IC50)比較二維培養與三維培養毒殺效果，在24小時分別為8.57 μg/mL 與13.63 μg/mL，而在48小時分別為0.24 μg/mL與4.70 μg/mL。本研究製備出填充天然高分子支架之三維微流道並在倒立式顯微鏡載台安裝的二氧化碳細胞培養系統中成功培養人類乳癌細胞，並使用DMC100F藥物造成有效的毒殺效果。

關鍵字

甲基丙烯酸環氧丙酯改質玻尿酸 ； 艾黴素 ； 微影製程 ； 微流道晶片 ； 膠原蛋白 ； 藥物篩選

並列摘要

In this study, a microfluidic perfusion system was fabricated for three-dimensional culture of human breast cancer cell line (wild type MCF-7). The microfluidics made of polydimethyle siloxane (PDMS) elastomer was fabricated by lithography technology in which two layers of micro-channel were adhered on a glass substrate of 7.5 x 2.5 cm2. Porcine skin extracted type I collagen and UV-curable hyaluronic acid (HA having 21% grafted with glycidyl methacrylate) were filled in the bottom layer of the micro-chaneel and subsequently, UV cross linked to give a three-dimensional (3D) interpenertrating netweok. The sterilization of the microfluidic channels was achieved by flowing through 75% alcohol and UV irradiation (365 nm). After the sterilization, MCF-7 cells were statically incubated for 24 hours for cell adhesion and followed by perfusion of culture medium MEM at a flow rate of 8 μL/min. The microfluidic perfusion system, maintained at 37 oC, was mounted on inverted optical microscope. To avoid hypoxia in the micro-channel, Hypoxyprobe™-1 Plus kit was employed to determined whether the micro-channel was under a condition of hypoxia and a cell viability of 65% was determined by Lactose Dehydrogenase? (LDH) for an incubation time of 120 hours. Doxorubicin (Dox) loaded in four arms PEG-PCL polymeric micelles were prepared. The average particle size of the DOX-loaded micelle was 132.1 nm, and the drug loading efficiency were 87.1%, respectively. The total releases of DOX in a period of 60 hours at pH 7.4 and pH 4.9 were 21.3 % and 78.6 % of total Dox concentration loaded, respectively. The half lethal dose (IC50 ) of 2D (10-cm dish) and 3D microfluidic cell culture system were 0.24 μg/mL and 4.70 μg/mL at 48th hour, respectively. In conclusion, a microfluidic perfusion system for MCF-7 cells was developed and cell viability and sterilization were evaluated. Dox-loaded polymeric micelles was evaluated as model dosage form for drug screening applications.

並列關鍵字

Drug screening ； Lithography ； Microfluidics ； Collagen ； Glycidyl methacrylate hyaluronic acid ； Doxorubicin

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