以正篩選偵測稀少細胞之碟片微流系統

Abstract

在臨床研究中，血液中的特定稀少細胞可以當作醫生診斷治療病人的參考資料，例如：病情診斷、治療過程的監視、評估治療效果。而稀少細胞在血液中的濃度大約為十億分之一(ppb)，因此如何快速且廉價的分離和偵測血液中稀少細胞為醫學上很重要的挑戰。 本研究提供一個具有微流體結構的碟片設計，利用免疫磁力分離法和離心力來完成稀少細胞分離，並透過螢光顯微鏡來辨識細胞。但由於病人的血液取得不易，前期研究使用細胞株進行實驗，以人類乳腺癌細胞株(MCF7)模擬血液中之稀少癌細胞，及淋巴球癌細胞細胞株(Jurkat Clone E6-1,簡稱：Jurkat)模擬血液中之白血球(表示血液中的大量的細胞)。MCF7與Anti-CD326-PE 和 Anti-PE BD magnetic beads 進行結合，使其能被磁鐵所抓取，並發出紅色螢光。Calcein-AM則用來標定Jurkat，使其發出綠色螢光。 將少量MCF7和大量Jurkat混合成300μl的細胞混合液，並注入微流體碟片中。接著放上多環磁鐵，靜置兩分鐘。經過旋轉碟片，MCF7被磁力留在內圈，Jurkat 和溶液被離心力排除到碟片外圓。最後利用螢光顯微鏡直接計算碟片上被分離的MCF數量。 實驗結果顯示，在跨越三個不同數量級的Jurkat (106、107、108)中，各加入少量的MCF7 (10+、100+、300+) 混合，均有 80%的MCF7可以在微流體碟片中穩定地被檢測到。證明此微流體碟片具有穩定的分離效果和良好的靈敏度。整個實驗過程包括細胞分離和細胞偵測，所需要的時間大約是30分鐘。細胞分離過程較簡單、低成本，而細胞損失仍然在可接受的範圍內。所以這個微流碟片將可以用來偵測目前有明顯特定免疫性質的細胞。例如：偵測癌症病人血液中的癌細胞(簡稱：CTCs)、母親血液中的胎兒細胞等。多了這些診斷資料，醫生可以為病人量身訂做適合的治療方法，並減少檢查過程中的風險。

In clinical studies, concentration of specific rare cells in the blood can serve a variety of purposes, e.g. disease diagnostics, evaluation of treatment efficacy, and disease prognosis. With the ratio of rare cells to whole blood cells in the range of one per billion (ppb), detection and separation of rare cells from whole blood via quick and inexpensive methods present important challenges in medicine. This thesis presents a proof-of-concept study via a compact disk (CD)-based microfluidic platform to separate and detect rare cells in blood. The disk platform utilized immuno-beads and a multi-stage magnet to separate rare cells. Detection of rare cells was performed in the disk with. The transfer force of liquid translation was centrifugal force via adjusting the rotational speed. The blood of patients was inconveniently obtained, so the cell lines were used in the preliminary experiments. The MCF7 were used to simulate the circulating tumor cells (CTCs) as target and stained with anti-CD326-PE antibodies and anti-PE BD magnetic beads. Jurkat Clone E6-1 was used to simulate the large number of cells in human blood, such as leukocyte, erythrocyte, and other cells. In order to identify with MCF7, Jurkat were labeled with Calcein-AM. The 300μl cell mixture containing few MCF7 and great number of Jurkat was injected into the microfluidic disk. And then the multi-stage magnet was put at the top of disk for two minutes. After rotation, the MCF7 was captured at the top of inlet reservoir, and Jurkat and solution were depleted to outer rim. The captured MCF7 in mircofluidic disk were detected with a fluorescence microscope. Results indicate that while small amount of MCF7 cells (10+、100+、300+) mixed into three different amount of Jurkat cells (106, 107, 108), the yield of detecting MCF7 were approximately 80%. The yield of MCF7 is independent of the number of Jurkat, and the microfluidic disk has well sensitivity (one in 107). The compact disk performs separation and enumeration of rare cells within roughly 30 minutes with relative ease and low cost at an acceptable level of cell loss. The microfluidic disk should be readily applied to separate and detect many rare cells with distinct immunological features, such as CTCs in cancer patients’ blood, the fetal cells in maternal blood, and endothelial cells in human blood. According to more information of diagnosis, doctor would make the personal treatment for patients. And the microfluidic disk could reduce the risk of assays.