微流體技術探討腫瘤微環境對誘發上皮至間質細胞轉換及癌幹細胞之研究

Abstract

近年來，針對單顆細胞或是極少數量細胞的研究主題越來越受到重視，特別是應用於幹細胞治療、人體內循環稀少細胞研究以及僅需使用微量的試劑來針對這些細胞做藥物篩選及開發等。再者，生物發展是一個非常複雜的過程，因此更需要開發一個新技術來精準地操控這些稀少細胞，並建立一個可模擬體內微小環境的生物反應器。 本論文為了達到以上所提及的研究目的，因此開發出一個微流體晶片，該晶片可精準地抓取單顆細胞並可直接在晶片內培養以及模擬體內微環境的一些特徵變化。此晶片包含上和下流道各一，中間夾著一層多孔性薄膜，單顆細胞可被準確並快速地排列在孔洞薄膜上，其中的操作程序僅需要手動微調上下流道的水位差壓力即可達到，完全不需要額外複雜的幫浦系統來推動。再者，該晶片的細胞捕捉率可達到97%；透過細胞外基質以及仿生奈米纖毛來選擇修飾薄膜表面、捕捉並培養人類癌細胞，我們的初步實驗結果顯示出該晶片可成功地實現二維(2D)細胞排列以及均勻分佈的三維(3D)細胞球培養。此外，三維細胞培養已被證實更近似於人體內的微環境，又癌症轉移過程跟細胞-間質間轉換 (epithelial-mesenchymal transition; EMT)有密切的關聯，因此本論文也將針對這個現象做更深入的探討。從實驗結果中我們發現利用奈米纖毛開發的三維細胞培養平台可用來觀察動態及可反向運行的外皮至間質細胞轉換過程。此外，奈米纖毛可促進癌細胞獲得類似癌幹細胞的表現特徵以及對化療藥物的抗藥性，再者，我們也發現三維細胞球培養出的癌細胞具有較高的轉移能力。最後，本論文所開發的微流體晶片以及細胞培養平台不但具有可主動操控二維和三維細胞排列及培養的優勢，並且於未來可直接被應用於探索研究極為重要的抗癌藥物開發以及癌幹細胞發展過程。

Study on single cells, or small number of cells, are important for many reasons. Among them, the drive towards stem cell therapy, study of clinically important rare cells, and the need to reduce reagent cost in cellular screening process all contributed to recent upsurge in single cell activities. Moreover, biological processes are often so complicated that the ability to provide insight in controlled cellular microenvironment using single or small number of cells can be extremely insightful. This work aims to design a microfluidic chip and three-dimensional (3D) culture platform capable of single cells and tumor microenvironmental studies. Specifically, the microchip allows mimicking the in vivo microenvironment to recapitulate the physiological conditions, which is known to be of utmost importance. The microfluidic chip consists of an upper and a lower chamber sandwiched by a thin perforated membrane patterned with 10-μm openings. Cells are transported hydrodynamically in the top channel of the two-channel structure via pressure gradient along the top channel. Suitable pressure difference across the membrane (via the through-holes) is achieved by fine-tuning the pressure gradient across the membrane. As the cells approach the through-holes, this pressure difference immobilizes the cells onto the membrane, achieving desirable cell patterning. Results show the chip is capable of deterministic patterning of cells in 2D or 3D by bioengineering the cell-supporting membrane both using extracellular matrix (ECM) molecules and biomimetic nano-cilia (triblock copolymers). The cell trapping rate attains 97%. Tuning of the surface enables not only highly controlled geometry of the monolayer (2D) cell mass but also 3D culture of uniformly-sized multicellular spheroids. This microchip will be interrogated to study both 2D and 3D cellular patterns using culture of human epithelial cancer cells. Of particular interest, the process of metastasis believed to be closely related to epithelial-mesenchymal transition (EMT) will be studied in detail. The dynamic and reversible regulation of EMT is examined in spheroids passaged and cultured in copolymer-based platform. The expression of CSC markers, including CD44, CD133, and ABCG2, and hypoxia signature, HIF-1a, is significantly upregulated compared to that without the nano-cilia. In addition, these spheroids exhibit chemotherapeutic resistance in vitro and acquired enhanced metastatic propensity, as verified from microfluidic chemotaxis assay designed to replicate in vivo-like metastasis. In conclusion, the proposed microfluidic chip and nano-cilia-based culture platform not only may offer new opportunities to achieve active control of 2D cellular patterns and 3D multicellular spheroids on demand, but may be amenable towards general study of important processes involving anti-cancer therapeutics and cancer stem cells (CSCs) in vitro.