微流道裝置中進行目標細胞之電穿孔

Target Cell Electroporation in Microfluidic Device

10.6342/NTU201603473

黃聖凱

基因治療 ； 電穿孔 ； 微流道 ； 細胞抓取 ； 橫向流 ； 轉染腺病毒E1A基因的人腎上皮細胞系 ； gene therapy ； electroporation ； microfluidics ； cells isolation ； cross flow ； HEK 293T cells

國立臺灣大學生物產業機電工程學研究所學位論文

2016年

碩士

盧彥文

英文

在在現在的細胞治療及基因轉殖的技術上，主要的關鍵在於如何能夠突破細胞膜的限制，把基因或藥物送入，藉此得知單一種分子對細胞的影響。在傳統電穿孔儀器上，所需要的電壓、樣品量及產生的廢熱都會比較高，且對象只能針對大量細胞，分子對細胞的影響只能藉由表現的平均值的方式去推算，無法針對單一細胞進行了解。 因此本研究致力於發展微流道裝置進行單一細胞之電穿孔，實驗以轉染腺病毒E1A基因的人腎上皮細胞系(HEK 293T cell)為例，利用流體集中(hydrodynamic focusing)的方法配合橫向流(cross flow)的壓力將目標細胞推送至抓取L型結構陣列中。接著對結構的兩側的ITO電極施加不同的脈衝電壓，造成細胞膜破洞，執行電穿孔，同時將螢光染劑及pEGPF-c1螢光蛋白送入細胞，以利觀察，驗證電穿孔的結果。本研究所設計的微流道裝置成功的達到針對單一目標細胞電穿孔的效果。

One of the key techniques in gene delivery and cell therapy is to break through the limit of the cell membrane and to send gene or drug into the cell. Electroporation is one of such methods. However, it conventionally suffers the drawbacks in high voltage, large sample amount and high heat generation. The gene/drug delivery results can be only estimated for the average gene expression of the group of the cells, instead of a single cell. To realize a particular gene expression onto a single cell, our thesis thus is devoted to perform electroporation on a single cell while to improve the limitation of the traditional electroporation techniques. A microfluidic device for single cell electroporation was presented. The HEK 293T cells were tested. The cells were first confined at the L-shape trapping structure with cross-flow cell filters enhanced by hydrodynamic focusing forces. Once the cells were trapped, the different voltage were applied for electroporation. Fluorescence dye and pEGFP-c1 plasmid were successfully delivered into the cells to validate the devices.

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