利用微流體系統產生之微液珠提升Lentivirus感染自然殺手細胞效率之研究

Abstract

免疫細胞療法可以被廣義的定義為疾病的治療方法，而近年來免疫細胞療法漸漸被視為治療癌症的革命性方式。本研究將Lentivirus（慢病毒）與自然殺手細胞的傳統實驗室進行方式，結合微流道晶片技術，進行細胞活性與 DNA 表現與慢病毒感染自然殺手細胞的的測試與實驗，使用DNA活性染劑：（7-AAD 7-氨基放線菌素D、7-Aminoactinomycin D）染細胞的DNA與利用其表現的綠色螢光蛋白（ Green fluorescent protein, GFP）來分析旨在改善其良率。 而在應用層面上，本研究中的微流道晶片，為 了讓細胞表現特定蛋白質，慢病毒載體基因傳遞需要進入有興趣的細胞，透過在微流道晶片的結構設計，讓慢病毒和自然殺手細胞先接觸，讓病毒將特定的DNA帶入自然殺手細胞的細胞核內，沿著流場方向流動，由連續相－油一起被包裹在液滴內，此時大量增加了彼此接觸的表面積、擴散距離縮短，達到單位體積內病毒濃度增加和實驗時間減少的目的。微流道材質為PMMA（聚甲基丙烯酸甲酯），由雷射雕刻機和熱壓機製造晶片，結 構設計採用聚焦流動的型式生成液滴，減少液滴與壁面的接觸，能避免考慮壁面疏水性的問題，維持實驗的穩定與重複性。 流式細胞儀（flow cytometer）被廣泛的應用於生物的醫學研究與分析，可以分析、紀錄流動狀態的細胞和發射螢光信號，用於分析細胞的活性、顆粒狀態和反映其生物學特性等。本研究於點狀分析圖（Dot Plot）中觀測細胞的活性與表現的綠 色螢光蛋白分佈，實驗在經過微流道晶片後，持續培養若干小時後，觀測其活性與感染狀況 。

Immune cell therapy can be broadly defined as a treatment method for diseases. In recent years, immune cell therapy has gradually been regarded as a revolutionary way to treat cancer. This study uses the traditional laboratory method of lentivirus and natural killer cells, combined with microfluidic chip technology, were used to test and experiment on cell viability and DNA performance and natural killer cell infection by lentivirus, using DNA active dye: 7-AAD (7-Aminoactinomycin D), the DNA of cells stained with the expression of Green fluorescent protein (GFP) is used to analyze, in order to improve its yield. The application of this research, in order to allow cells to express specific proteins, the lentiviral vector gene delivery needs to enter the cells of interest. Through the structural design of the microfluidic chip, the lentivirus and nature killer cells contact first, let the virus bring specific DNA into the nucleus of the nature killer cell, and flow along the direction of the flow field. The continuous phase and oil are wrapped together in the droplet. At this time, the contact surface area is greatly increased and the diffusion distance is shortened. , To achieve the purpose of increasing the virus concentration per unit volume and reducing the experimental time. The material of the micro flow channel is PMMA (polymethyl methacrylate). The wafer is manufactured by a laser engraving machine and a hot press. The structure design adopts a focused flow pattern to generate droplets, which reduces the contact between droplets and the wall and avoids considering the wall The problem of hydrophobicity maintains the stability and repeatability of the experiment. Flow cytometer is widely used in biological medical research and analysis. It can analyze and record cells in the flow state and emit fluorescent signals, and is used to IV analyze cell activity, particle state and reflect its biological characteristics, etc. In this study, the cell activity and the expression of the green fluorescent protein distribution were observed in the dot plot. After passing through the microfluidic chip system, the experiment continued to incubate for several hours to observe its activity and infection situation.