人類初代自然殺手細胞毒殺特性與即時分析之微流體晶片研究

Cell-based Microfluidic Device for Cytotoxicity and Real-time Investigation of Primary Human Natural Killer Cells against Leukemic Cells

林羿彣(Yi-Wen Lin)

指導教授 ： 黃榮山

國立臺灣大學/工學院/應用力學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.02849

摘要

自然殺手細胞已成為抵抗癌症中細胞治療之注入細胞之一，在癌症療法中，細胞治療的副作用低，且能延長病患壽命並有效改善生活品質。由於來自不同捐贈者的自然殺手細胞呈現相異的毒殺癌細胞效果，選擇適當且高毒殺效果的捐贈者，是一重要課題。再者，傳統上用於測試細胞毒殺率的方法必須使用大量細胞(2×105細胞數)作毒殺測試，再用昂貴的流式細胞儀作分析與統計。在臨床應用上，不僅不利於取得珍貴、稀少且有限的捐贈者細胞挪出高比例的細胞量作毒殺測試，而且昂貴的流式細胞儀不利於分析的普及性。
因此，本研究提出一細胞晶片，成功地使用臨床實務之初代自然殺手細胞為作用細胞，不僅利用少量細胞(~101細胞數)，針對血癌細胞株K562作毒殺測試，也以此細胞晶片作分析，以取代昂貴且需大量細胞之流式細胞分析儀之功能，成功地作為少量細胞毒殺測試之即時分析平台。再者，本研究也採用了臨床實務上，測試與分析來自三位不同健康捐贈者初代自然殺手細胞之毒殺效率，其結果可提供合適的捐贈者作為細胞治療植入的依據。
本研究以微機電技術製造針對懸浮細胞之晶片，並整合光學觀測系統。配合微流體操控技術與間隙結構，可捕捉約數十顆(~101細胞數)的細胞樣本，計算適當的微流體對細胞作用力以維持細胞活性，並完成細胞凋亡染色的確認，高解析度光學與即時影像觀察系統完整紀錄反應過程。透過傳統與微分析平台之比較，兩者毒殺率的分析誤差在6%以內，證明此細胞晶片可成功取代傳統多量細胞作毒殺測試與流式細胞儀作分析之實驗方式。其中，來自臨床上三個健康捐贈者之自然殺手細胞，將細胞經過體外培養連續三週，並每隔一週以相同代數的癌細胞株測試毒殺效果，結果三組自然殺手細胞的毒殺效果皆在體外培養後14天即無顯著成長，毒殺率分別是：70.07%、66.53%以及61.01%，此結果驗證本晶片在臨床的應用上，可挑選效果較佳的捐贈者。本晶片在分析毒殺率的同時，可透過即時影像紀錄毒殺過程，有別於過去影像的觀測和毒殺效果分析分別建立在兩套不同系統上，此晶片更有益於幫助探討自然殺手細胞之基礎特性。本研究可成功地利用臨床實務之人體初代殺手細胞，利用少量細胞作毒殺測試與即時分析，並可作為挑選捐贈者的測試分析，對臨床治療癌症的即時性具應用價值。

關鍵字

人類自然殺手細胞 ； 微流體 ； 生醫晶片 ； 微機電製程 ； 即時觀測系統

並列摘要

Natural killer cell transplantation therapy is potential to cancer treatment. It is able to killer cancer cells directly and has no need of haploidentical matched donor before transplantation. Since natural killer cells from different donors perform different cytotoxicity against cancer cells, it is significant to choose an appropriate donor who is suitable for each patient. However, the conventional method for determination of cytotoxicity took large amounts of cells and could not provide image evidence of cell-cell interaction process. As a result, we developed a new appropriate assessment to select multiple healthy donors.
In this study, we designed a cell-based microfluidic device which was capable to integrate a real-time observation system. The effector cells we used were primary natural killer cells isolated from peripheral blood. The target cells were leukemic cell line K562. The cytotoxic assay performed by micro device needed only small amount of samples. And the results showed that the micro device is able to replace conventional cytometry to determine the cytotoxicity of natural killer cell. Furthermore, this study adopted primary natural killer cells from three healthy donors. Therefore, the experimental results provide the basis for a suitable donor in transplantation.
This study developed a micro device applied on research of suspension cells. The micro device was fabricated by MEMS technique and easily integrated with real-time observation system. With microfluidic manipulation and the designed gap structure, the micro device could trap dozens of cells. The liquid pressure difference between central and side channels were controlled to maintain cell docked in the gap and complete cell identification of apoptosis. The cytotoxicity results from the micro device were consistent with that from conventional cytometry. Moreover, the cytotoxicity of NK cells from donor A, B and C which were cultured in vitro for 14 days were 70.07%,66.53% and 61.01%, respectively. After culturing 21 days in vitro, the cytotoxicity of NK cells were not growing significantly. We proved that the cell-based biosensor reduced the consumption of cell sample from the number of 2×105 to 101 for detection and present the capability and potential for application of natural killer cell transplantation.

並列關鍵字

natural killer cell ； microfluidic ； biosensor ； MEMS ； real-time observation

參考文獻

[1] International Agency for Research on Cancer. Available: http://www.iarc.fr

[2] L. Ruggeri, A. Mancusi, K. Perruccio, E. Burchielli, M. F. Martelli, and A. Velardi, "Natural Killer Cell Alloreactivity for Leukemia Therapy," Journal of Immunotherapy, vol. 28, pp. 175-182, 2005.

[3] J. E. Rubnitz, H. Inaba, R. C. Ribeiro, S. Pounds, B. Rooney, T. Bell, C.-H. Pui, and W. Leung, "NKAML: A Pilot Study to Determine the Safety and Feasibility of Haploidentical Natural Killer Cell Transplantation in Childhood Acute Myeloid Leukemia," Journal of Clinical Oncology, vol. 28, pp. 955-959, February 20, 2010.

[4] L. Ruggeri, A. Mancusi, M. Capanni, E. Urbani, A. Carotti, T. Aloisi, M. Stern, D. Pende, K. Perruccio, E. Burchielli, F. Topini, E. Bianchi, F. Aversa, M. F. Martelli, and A. Velardi, "Donor Natural Killer Cell Allorecognition of Missing Self in Haploidentical Hematopoietic Transplantation for Acute Myeloid Leukemia: Challenging Its Predictive Value," Blood, vol. 110, pp. 433-440, July 1, 2007.

[6] K. Guldevall, B. Vanherberghen, T. Frisk, J. Hurtig, A. E. Christakou, O. Manneberg, S. Lindstrom, H. Andersson-Svahn, M. Wiklund, and B. Onfelt, "Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays," PLoS ONE, vol. 5, p. e15453, 2010.