本研究成功利用微流體技術建構出微型三維細胞培養平台，並將此平台運用於測試抗癌藥物對癌細胞之影響。此三維細胞培養平台特色便是能夠提供穩定且均一之細胞培養環境，且於此平台中，發展出一種細胞/膠體注入機制，能夠將已先混合好之細胞/膠體快速且簡易注入所有細胞培養區當中且不會阻塞到其他微流道中培養液之流動，以達到高通量化三維細胞培養之目的。而此三維細胞培養平台之晶片製程步驟是以SU-8製程先建構出此平台各層之母模結構，再利用具有高生物相容性且具有良好透光性之聚二甲基矽氧烷 (PDMS: poly-dimethylsiloxane) 翻模。而在於此微型三維細胞培養平台當中，大幅減小了三維細胞培養規格尺度之大小，不僅達到減少實驗資源之消耗，更成功解決了存在於一般三維細胞培養物中之化學梯度問題。甚者，成功整合了兩種微型蠕動式幫浦於此細胞培養平台當中(包含 “蜘蛛網式微型蠕動式幫浦” 以及 “利用氣體分流機制與新式S型蠕動式幫浦” )用於細胞培養液之連續輸送，以取代傳統式之外加大型流體輸送設備，達到經濟化之效果。而發展出之細胞/膠體注入機制，更能夠大幅減少人為上操作造成各個細胞培養區中細胞膠質基注入量不同之誤差。而在於實驗結果中，顯而易見地表明出此15組微型蠕動式幫浦在於三維細胞培養平台中，能夠提供各個培養液輸送管道之流速均一性 (其流速值大小範圍(取決於外加電磁閥所給與的頻率): “蜘蛛網式微型蠕動式幫浦”輸送系統是3.6 ~ 439.0 微升/小時。“利用氣體分流機制與新式S型蠕動式幫浦” 輸送系統是5.5 ~ 131微升/小時)。而在於細胞膠質基注入機制方面，能夠利用不同壓力值施加於微型閥門上用來控制細胞/膠體注入之體積量，因此實驗結果表示當給與微型閥門壓力值為10、12、15磅/平方英吋時，便能夠得到相對應之細胞/膠體之注入體積量分別是0.22、0.18、 0.14 微升。而最後，亦將此培養平台用於人類口腔癌細胞之培養及抗癌藥物測試。

This study reports a new perfusion-based, micro three-dimensional (3-D) cell culture platform for drug testing using enabling microfluidic technologies. The platform provides the features of homogenous and well-defined culture environments, efficient medium delivery and “smart cells/ agarose (scaffold) loading mechanism”, allowing more precise and high-throughput 3-D cell culture-based assay or testing to be realized. In this work, the perfusion-based, micro 3-D cell culture platform is designed and is fabricated based on SU-8 lithography and PDMS (poly-dimethylsiloxane) replication processes. The miniaturization of the culture scale in the culture platform contributes favorably to a low chemical gradient culture environment. Also, two types of pneumatic-based medium pumping mechanisms (the “spider-web” peristaltic pneumatic micropumps system and “modified serpentine-shape” pneumatic micropump system associated with an air tank) were integrated into the micro 3-D cell culture platform to provide efficient and economical culture medium delivery mechanism. Moreover, the “smart cell/agarose (scaffold) loading mechanism” was proposed, allowing the cells/3-D scaffold loading process in one step and avoiding too much laborious works and error caused by manual loading. The results show that in all of the 15 pneumatic micropumps studied, the medium delivery mechanism is able to provide a uniform flow output ranging from 3.6 to 439.0 µl/hr for the “spider-web pneumatic micropump system” and 5.5 to 131 µl/hr for the “modified serpentine-shape” pneumatic micropump system associated with an air tank, respectively, depending on the applied pulsation frequency of the micropumps. In addition, the cell/agarose (scaffold) loading mechanism is proven to be able to perform sample loading tasks precisely and accurately in all of the 15 microbioreactors investigated with adjustable loading volumes of 0.22, 0.18 and 0.14 µl at applied air pressures of 10, 12 and 15 psi, respectively, in the microvalves. Furthermore, anti-cancer drug testing is successfully demonstrated using the proposed culture platform and fluorescent microscopic observation. As a whole, because of miniaturization, not only does this perfusion 3-D cell culture platform provide a homogenous and steady cell culture environment, but it also reduces the need for human intervention. Moreover, due to the integrated pumping of the medium and the cell/agarose (scaffold) loading mechanisms, time efficient and economical research work can be achieved. These characteristics are found particularly useful for high-precision and high-throughput 3-D cell culture-based drug testing.

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