利用微流體技術建立三微細胞團培養系統探討細胞分化之研究與癌症藥物測試

Developments of Three-dimensional Multicellular Spheroid Culture Systems for Cell Differentiation Studies and Anti-cancer Drug Testing Using Microfluidic Techniques

傅芊瑜(Fu, Chien Yu)

指導教授 ： 張晃猷

國立清華大學/生命科學院/分子醫學研究所/博士(2015年)

摘要

多細胞球體由於其結構與型態和真實的組織極其相似，經常被選擇作為體外三維細胞培養模式。此外，因提供適當的細胞與細胞及細胞與細胞基質間交互作用，在維持體外培養之初代細胞的特定功能及調節幹細胞分化潛力上亦有顯著幫助，目前已廣泛應用在基礎生物及藥物篩選之相關實驗。因而，在醫藥生物研究中，建立合適之細胞球體模式並有效率的進行培養與觀察方法成為球體技術持續發展上重要的一環。近十幾年來發展的微流體技術具備精準操控流體的能力，加上與其他工程技術結合製成的實驗室晶片，能夠在體外建立接近體內的生物微環境來進行細胞培養及實驗分析，藉此使科學家們獲取更可信的研究結果。在本論文中，便針對多細胞球體相關的研究及當前發展遭遇的問題進行微流體晶片系統的設計開發與測試。首先，我們在微流體系統中設計特定微結構進行對單一個多細胞球體的不同部位進行不同的細胞分化，培養由多種類的細胞形成的多細胞球體。這類異種種類細胞形成之多細胞球體能作為更複雜的三維模式以研究多種類細胞間的交互作用亦或是應用在組織工程領域。接著，我們建立以微流體晶片培養多細胞球體進行抗癌藥物篩選的平台，藉著微流體晶片內製作之Ｕ型光敏水膠微結構進行細胞捕捉、多細胞球體形成與培養以及處理抗癌藥物後的細胞毒殺測試。隨著三維多細胞球體在基礎研究與藥物發展上的應用與重要性逐漸提升，我們相信結合微流體技術進行多細胞球體之培養與分析將帶給此領域更多研究過程的便利性, 更接近細胞於生物體內的行為, 以及發展未來快速高通量藥物篩選及自動化實驗的想法。

關鍵字

多細胞團塊 ； 微流體 ； 三微細胞培養 ； 藥物篩選

並列摘要

Because of their structural and functional resemblance to real tissues, multicellular spheroids have been widely used as 3-D tissue models for basic cell biology studies and cell-based drug screenings. Spheroids have also been implicated as a powerful method to maintain or enhance cellular functions in primary cells and regulating the potency of stem cells. Recent advancement of microfluidic technology has provided scientists many capabilities including continuous dynamic perfusion and automatic fluidic control in performing cell culture. These functions are beneficial for growing cells in a long-term culture and their subsequent treatment and analysis. In this thesis, we first designed microfluidic systems to control cell differentiation at a specific site on a single spheroid. These strategies could controllably generate heterospheroids with a desired arrangement of multiple cell types, which meets the demand of a more complex MCS-based model for tissue surrogates for application in tissue engineering and cell-cell interaction studies. Furthermore, for the establishment of a well-manipulated process for spheroid productions and spheroid-based tests, we also generated a microfluidic device integrated with U-shaped PEG hydrogel microstructures that allow cell trapping, spheroid formation through self-assembly, and long-term culture of the spheroids. Heterospheroids generations and tests of anti-cancer drug on tumor spheroids have been accomplished and are potentially for automation through this microfluidic system. To summarize, we believe that applying the microfluidic techniques for establishment of desired 3D cell culture systems is beneficial for fundamental biological studies, drug-screening and other pharmaceutical application in the future.

並列關鍵字

multicellular spheroids ； microfluidic ； 3D cell culture ； drug-screening

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