建構高通量慣性力分離之微流體晶片應用於全血中之白細胞篩選

人體的某些細胞有時會因為失去正常的生理控制機轉而快速生長，造成腫瘤。這些細胞自行增長，自成一群集團，無視於身體的正常需求，也不週遭正常組織的規範，它們漫無目的變大，終至影響到人體的功能。通常這些生命單位依照預期正常的速度分裂，取代舊有的細胞。而當細胞變成癌細胞時，細胞其實是發生了很危險的變化，特別是細胞核內的DNA。正常情況下，當細胞的DNA出問題時人體內的免疫系統會啟動對付這些癌細胞。但若不幸地失靈，這些有問題的DNA就會產生瘋狂分裂的現象。而癌細胞的轉移有許多方式，其中一種是經由血液循環，癌細胞在原病灶放出血管增生素，使血管朝癌細胞生長，進入血液循環後又用血小板把自己包起來，降低被免疫系統發現的機會，直到最後卡在微血管，進而發展出第二病灶。在轉移過程中，大部份癌細胞被白血球消滅，但在成千上萬的癌細胞中只要有一個存活下來到達血液循環處，就會形成轉移。而經研究指出，進入血液循環之循環癌細胞數量，對於病患的治癒率及死亡率有指標性意義，因此從血液中分離或捕捉循環癌細胞成為重要幫助特別在分子癌症學及免疫省理學裡而且具有極大意義的議題。而目前的生物晶片發展技術並沒有很有效及快速的對血液裡的血球進行篩選。主要原因是其對象(白血球或癌細胞)與紅血球在血液內含量相差將近1000倍而且各種血球在微流體晶片在同時大量進行分離或篩選時(4.21x106 /μl)造成更容易阻塞等等問題。因此，本研究利用黃光微影製作出微流體生物晶片純粹利用流體動力就能足夠對全血血液裡有效的濾除紅血球有易於後續對白血球及癌細胞檢驗。考慮到血液內大多數的紅血球與白血球各具有不同的大小及形狀之彈性力造成在流體裡走的路徑也有所改變，特別是紅血球在流體內流動時受到剪變率及黏質力之關係而聚焦在傾向微流道的內壁。藉由此流體現象而晶片微結構設計出一個對稱性的彎道，再加上沿著彎曲流道內壁設成曲率半徑之陣列的間隔(7μm)而這間隔將通到另外出水槽。這刺激間隔設為7μm的寬度除了紅血球碗狀結構及其直徑約7-8μm而目標檢測的對象大小約15μm。因此，在測試晶片的效率時會使用泵浦抽取方式能讓血液在進入流道時大部分的紅血球被吸進刺激結構裡而白血球或癌細胞能維持在主流道。另外，本實驗所採用的晶片是高分子材料(PDMS)，除了生物相容性高，適合於醫學檢測；且其價格較低，可大量生產製造，將有效降低以往檢測設備成本。目前測試最佳的篩選流體速度為0.6 c.c /min. 等於在50分鐘內將1c.c的血液分離完。雖然已成功把白血球分離出 95% 的白血球以及4.9%的紅血球 (相當於3.3 × 109 細胞/mL)但為了能夠運用在循環癌細胞早期檢測上，此結果還是須要一些改良提高細胞分離效率至少能分出95% 以上白血球之純度。而且這實際上的血球及癌細胞數量相比109 顆 : 5-20顆癌細胞比例跟這些初步結果差距還是相當的大。

關鍵字

慣性力；血液；白細胞

並列摘要

Presently, viable tumour-derived epithelial cells (circulating tumour cells or CTCs) have been identified in peripheral blood from cancer patients and are probably the origin of intractable metastatic disease. And the cells are extremely rare, tumor cells represent a potential alternative to invasive biopsies as a source of tumour tissue for the detection, characterization and monitoring of non-hematologic cancers. The ability to identify, isolate, propagate and molecularly characterize cancer cell subpopulations could further the discovery of cancer stem cell biomarkers and expand the understanding of the biology of metastasis. But the current strategies for isolating CTCs are limited to complex analytic approaches that generate very low yield and purity. In this work, we design a microfluidic chip based on inertial hydrodynamic force to help on the separation of CTC cells from normal cells. This in vitro cells separation device provides simple control, cheap and high throughput way to enrich CTCs based on the interaction of two majors hydrodynamics forces in flow. One is the wall repulsion force due to the steric crowding effect between the particle and the wall, and another is the inertial lift force that originates from the shear-gradient of the flow. The wall repulsion force pushes the particle away from the wall and the inertial lift force draws the particle toward the wall. Hence, the balance between these two oppositely directed forces induces an equilibrium position at a certain equilibrium position. The wall repulsion force pushes the particle away from the wall and the inertial lift force draws the particle toward the wall along the combination of several circulating straight, curve channel and suction flow of CTCs in huge number of whole blood cells. In the preliminary section we successful demonstrated the separation efficiency of erythrocytes (red blood cells) up to 95% means about 3.31 billion of ~5billion cells in milliliters has been sucked out into waste chamber and keeping the peripheral blood leukocytes (white blood cells) or circulating tumor cells in main channel with most of it are stacked at separation entrance or collection chamber. This current result extremely fast separation just less than 50 minutes and high enrichment device that have a great potential to replace recent clinical or biology cells detection and development technique such as, centrifugation with lysis buffer, electrophoresis separation chip, or even flow cytometer.

並列關鍵字

Inertia Force ； Blood ； White Blood Cell

參考文獻

【3】 Masumi Yamada, Megumi Nakashima, and Minoru Seki, “Pinched flow fractionation continuous size separation of particles utilizing a laminar flow profile in a pinched microchannel”, Anal. Chem. 2004, 76, 5465-5471

【4】 Junya Takagi, Masumi Yamada, Masahiro Yasuda and Minoru Seki, “Continuous particle separation in a microchannel having asymmetrically arranged Multiple Branches”, Lab Chip, 2005, 5, 778–784

【5】 Masumi Yamada and Minoru Seki, “Hydrodynamic filtration for on-chip particle concentration and classification utilizing microfluidics”, Lab Chip, 2005, 5, 1233–1239

【6】 Masumi Yamada, Kyoko Kano, Yukiko Tsuda, Jun Kobayashi, Masayuki Yamato, Minoru Seki and Teruo Okano, “Microfluidic devices for size-dependent separation of Liver Cells”, Biomed Microdevices (2007) 9:637–645

【7】 J. Park, K. Cho, C. Chung, D-C. Han, and J.K. Chang, “Continuous plasma separation form whole blood using microchannel geometry”, Proceedings of the 31 Annual International IEEE EMBS Special Topic Conference on Microtechnologies in Medicine and Biology Kahuku, Oahu, Hawaii 12 -15 May 2005

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建構高通量慣性力分離之微流體晶片應用於全血中之白細胞篩選

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