以明膠微圖案進行細胞之操控貼附、培養與監控

機構典藏 > College of Engineering > Graduate Institute & Department of Mechanical and Electro-Mechanical Engineering > Thesis > Item 987654321/35471

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Title:	以明膠微圖案進行細胞之操控貼附、培養與監控
Other Titles:	Application of gelatin micropatterns in cell attachment, culture and in-situ monitoring
Authors:	歐育誠;Ou, Yu-cheng
Contributors:	淡江大學機械與機電工程學系博士班楊龍杰;Yang, Lung-jieh
Keywords:	戊二醛;明膠;交聯;細胞培養;癌細胞;阻抗感測;即時監控;glutaraldehyde;gelatin;crosslink;cell culture;tumor cell;impedance sensing;in-situ monitoring
Date:	2009
Issue Date:	2010-01-11 06:37:42 (UTC+8)
Abstract:	本文提出一種新穎戊二醛交聯明膠之微成型技術，並且利用所成型之明膠微圖案來吸引癌細胞的生長貼附，除了以加入感光增感劑曝照紫外光(ultra-violet)交聯明膠方式與用戊二醛選擇性交聯方式之外，進一步利用氧氣電漿(O2 plasma)蝕刻方式來製作明膠微圖案，本新穎之明膠微圖案成型方式可以確保明膠之交聯程度、避免以戊二醛選擇性交聯方式所產生的過度交聯(over-crosslink)、增加明膠微圖案與玻璃基材之間的黏著程度，線寬解析度最佳可到達2微米，而利用傳統光蝕刻微影技術(photolithography)可以成功的製作出戊二醛交聯明膠之微圖案，利用明膠材料之特性，成功的吸引癌細胞產生選擇性之貼附生長。控制細胞生長的位置對於生醫感測器的製作、組織工程、生醫電子以及基礎生物研究而言是相當重要的，而本文則提供一個用以控制細胞生長位置的材料，也進一步將此材料實際應用於生醫感測器，監控細胞生長貼附狀態。在細胞培養過程中，細胞的黏著生長(cell adhesion)及其蔓延(cell spreading)是黏著型細胞的基本生長過程，其生長過程關係著人體的防禦系統、組織的形成與訊息傳遞路徑。而在個體細胞層級中，細胞的黏著生長(cell adhesion)與蔓延(cell spreading)關聯著單一細胞的許多特性，而單一細胞形態的改變，傳統上則使用光學的方式來進行量測，然而生醫感測器則提供了另一種量測方式。長久以來，電子細胞基質阻抗判斷技術(Electric Cell-substrate Impedance Sensing, ECIS)已經被認定是有效的方法，利用ECIS技術可以監測黏著型細胞之形態、活力以及細胞周遭環境變化。本文結合了ECIS技術、明膠微圖案成型技術與光學方式來製作一新穎之細胞培養系統，此系統包含一個具有即時觀察光學模組之培養箱以及整合明膠微圖案之ECIS感測晶片，此光學模組可以用於即時觀察培養箱中不同活體細胞目標，除了可透過光學模組觀察活體細胞活動，進行影像監控之外，接近細胞大小之小尺明膠微圖案直接被成型於工作電極上方，藉此明膠微圖案使細胞產生選擇性黏著，利用明膠微圖案控制細胞生長於感測電極上方，進一步透過感測晶片輸出之阻抗訊號變化，監控細胞生長貼附之狀態，同時透過光學觀察模組之即時影像也可確認量訊號變化來自細胞之不同生長貼附狀態。 This work proposes a novel technique to fabricate micropatterns of glutaraldehyde (GA)-crosslinked gelatin and induce the attachment of tumor cells to the gelatin micropatterns. It provides another method to crosslink gelatin other than using the photo-sensitizing agents or selective GA-crosslinked technique. The gelatin micropatterns are fabricated by O2 plasma etching process. This novel technique can ensure the degree of crosslink, prevent the over-crosslink from pattern deformation and enhance the adhesion between the gelatin and glass slide. The best spatial resolution of the gelatin micropatterns can be reached to 2μm. The micropatterns of GA-crosslinked gelatin can still be made successfully by the conventional photolithography. By the properties of gelatin, tumor cells are successfully attracted to produce a selective. It is important to control living cells onto the given surface for biosensor fabricating, tissue engineering, bioelectronics and basic biology studies. This thesis herein proposes a material to control cell positioning on the surface. Moreover, the material is actually employed in biosensor to monitor morphology of living cells. During the cell culture process, cell adhesion and cell spreading are fundamental processes of adherent cells. Furthermore, cell adhesion and cell spreading are also crucial to signal transduction pathways of a cell, the formation of tissues and the body’s defense system. At the individual cell level, some properties of a living cell are associated with cell adhesion and cell spreading processes. Traditionally, the changes of individual cell morphology are measured using the optical methods. In addition to the optical methods used to monitor the cell morphology of living cells, biosensors provide another means to monitor the changes of cell morphology. ECIS methods have long been regarded as a valid approach to monitor the morphology, viability, and environmental change of the adherent cells. This thesis combines ECIS technique, fabrication of gelatin micropattern and optical methods to set up a new cell culture system. This system includes a culture incubator with a compact optical real-time monitoring module and an ECIS sensor chip with gelatin micro patterns. The optical observing module is used for observing different targets of living cells in the incubator. Furthermore, the optical module can be used to monitor the cell behavior. In the meantime, the gelatin micropatterns fabricated firmly on the ECIS working electrodes have a small size which is purposely comparable to cells. By the gelatin micropatterns, cells are attracted to produce a selective adhesion during the stages of falling and attachment of cell culture. By this way, living cells can be controlled onto the given working electrode surface. The morphology of living cell can be monitored by changes of impedance signal from ECIS chip. Meanwhile, the data attributed to various cell morphologies also can be confirmed by the real-time image of the optical module.
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