手機螢光顯微鏡及雲端影像傳輸應用於微流體晶片中細胞毒殺之系統開發與研究

Abstract

近年來有越來越多的患者在免疫細胞治療中，選擇以自身免疫系統的自然殺手細胞體外增生，再注入體內毒殺血癌細胞治療白血病，免疫自然殺手細胞療法副作用小，在治療期間對病患來說相對其他傳統的化學療法有較良好的生活品質。在免疫細胞治療過程中，體外增生的免疫自然殺手細胞，注入體內前常需檢測其增生細胞之毒殺能力與活性，傳統上使用流式細胞儀分析細胞毒殺能力，需要大量的細胞數量(105~106)方能做可靠性的檢測，但對病患來說，增生後臨床用的自然殺手細胞數量有限、非常珍貴且昂貴，因此不適合挪用臨床用約百萬顆大量細胞作毒殺檢測之用。 本研究開發創新的光學系統裝置與細胞晶片，可作為僅需少量細胞（約102顆）之細胞毒殺檢測、即時觀測系統與即時雲端影像傳輸之智慧型手機螢光顯微系統裝置。本研究係利用(1)智慧型手機本身可做數位影像拍攝、自動對焦及雲端傳輸功能，(2)創新設計光學系統，類似倒立式螢光顯微光學之微型模組，包含LED光源、濾片及鏡片模組等裝置，(3)再利用微機電技術製作無幫浦微流體生醫晶片，藉由水柱高壓力驅動流體，配合微流道結構設計與微流體操控技術捕捉細胞，(4)以免疫細胞治療之自然殺手細胞與血癌細胞之毒殺作用並分析。整合以上四大項，以智慧型手機為基礎裝置，建構一個即時細胞檢測、螢光觀測與即時雲端影像傳輸之可攜式微型系統。 在微流體晶片，以二維矩形微流道中的流體水力動力聚焦理論模型，透過晶片中的管柱高度決定壓力與流速，產生水力聚焦現象，並設計微流道之間與間隙，透過流體壓力差，細胞會被吸引往間隙前進，由於細胞直徑(~10μm)大於縫隙高度(4μm)，因此細胞會被捕捉於主流道側壁的反應區，細胞間進行毒殺作用。 再者，創新之光學路徑設計、材料選擇，測試鏡頭的光學品質並計算其調制轉換函數約為0.2，最後透過光學元件的組裝並與無幫浦微流體生物晶片整合，結合成智慧型手機螢光顯微系統裝置與細胞晶片檢測，首先以螢光微珠作測試，測量此系統觀測視野區之光學特性並與傳統螢光顯微鏡影像比較，再進一步地測試真實的螢光染色細胞，可成功地測試螢光細胞流入微流體晶片。最後，以此創新之智慧型手機螢光顯微系統裝置與細胞晶片，利用自然殺手細胞及血癌細胞在此微型系統進行毒殺作用，以效能細胞（自然殺手細胞）對目標細胞（血癌細胞）比例為1：1的條件，在不同毒殺時間下，結果發現毒殺時間越長，其毒殺率也越高，並且將相同的細胞樣本以流式細胞儀進行分析比較，實驗結果具一致性。 本研究成功地以創新智慧型手機螢光顯微光學模組，取代傳統的螢光顯微鏡和微流體生物晶片，其功能並可取代傳統流式細胞儀之螢光細胞毒殺分析，此創新之細胞檢測系統，成功地運用少量細胞(~102)分析出細胞毒殺率，解決流式細胞儀需要大量細胞(~106)檢測的缺點，極適合臨床應用，其微型化攜帶方便且能即時遠端檢測，可應用於食品販賣現場檢測、實地環境檢測或科學教育之應用。

In recent years, a growing number of patients were cured by nature killer cell transplantation. Due to the little side effect of natural killer cells in cell transplantation, natural killer cell therapy has been chosen as an auxiliary strategy in post chemical treatment for good quality of life. Traditionally, cytotoxicity analysis is performed by using flow cytometry; however, it requires a large number of cells (~106). In clinical fields, proliferated nature killer cells are still limited and expensive, which remains a challenge for cytotoxic investigation by acquiring part of proliferated NK cells. In this study, the new smartphone-based fluorescence microscopy integrated with a microfluidic cell device was designed to be optically inverted by assembling highly bright LED, miniaturized lens module, and filter. Meanwhile, a microfluidic cell-trapped device was designed and manufactured by MEMS technique. With the design of microchannels, suspension nature killer cells and cancer cells were trapped by a fluidic pressure drop in microchannels. The miniature smartphone-based, inverted fluorescent microscopic system exhibited a real-time monitoring, cloud image computing, and cytotoxic analysis. In optics, optical quality and performance of the inverted smartphone-based fluorescence microscopy was characterized by area of interest, enlargement, and image resolution. The quality of lens was investigated by modulation transfer function (MTF) to be around 0.2 in MTF. In fluorescence characterization, fluorescent microbeads were used to characterize the area of interest in this microscopic system. The area of interest was found to be 2.5 mm2. At last, the cytotoxic analysis was performed by introducing nature killer cells and cancer cells into the microfluidic cell device. With the effector-to-target cell ratio of 1 between both cell groups, the cytotoxic result of the present system showed an excellent agreement with that in flow cytometer. This study successfully demonstrated the cell monitoring and investigation of the inverted smartphone-based fluorescence microscopy with the microfluidic cell device. In addition, the miniature integrated system presented the excellence in cytotoxic analysis in a small amount of cells (~102) in comparison with that in flow cytometry. With the integration of the smartphone and optical module, this new system can be wirelessly transmitted to the cloud for image computing and for remote detection. The present system creates a new platform of a portable, cost-effective inverted fluorescence microscopy, cell investigation, real-time field test with cloud image computing, and medical examination.