電漿表面改質對細胞培養塗層之研究

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Title:	電漿表面改質對細胞培養塗層之研究
Other Titles:	The Study of the Coating on Plasma Treated Surface for Cell Culture Coating
Authors:	戴緯 TAI, WEI
Contributors:	楊怡寬 YANG, YI-KUAN 化學工程與材料工程學系
Keywords:	電漿;表面改質;丙烯酸;架橋劑;膠原蛋白;細胞貼附 Plasma;Surface modification;Acrylic acid;Bridging agent;Collagen;Cell adhesion pattern
Date:	2019
Issue Date:	2019-12-16T02:18:48Z (UTC)
Abstract:	本研究主要是為了改善高分子材料表面的生物適應性，因此利用低溫真空電漿以氬氣與氧氣進行基材表面改質，使表面產生具有極性的基團如C＝O及－OH；選用的基材為通用的聚苯乙烯（Polystyrene）生物培養皿。本研究首先找到基材本身所能承受電漿能量的最大範圍與表面活性衰退的時間，在不破壞基材為前提之下找到表面活性最佳的時間、流量、瓦數等參數，並觀察其表面特性。之後我們將電漿處理過的基材以丙烯酸（Acrylic acid）水溶液加入起始劑（Ammonium persulfate）與抑制劑（Benzil α,α-dimethyl acetal）進行光接枝交聯，使其表面得到一個穩定且親水的丙烯酸薄膜來改善電漿處理後表面易隨著時間而衰退的問題。羧酸所組成的薄膜被認為對細胞的貼附生長有正面的影響，且丙烯酸本身的羧基可做為改質一個很重要的官能基；在接枝完丙烯酸後我們在低溫、酸性的環境下以EDC-NHS作為架橋劑將膠原蛋白（Collagen）固定化於丙烯酸的雙鍵上，製造出一個利於生物細胞生長的環境，而利用AFM做表面的粗糙度與機械性質的檢測，了解改質後表面結構的變化，透過ATR-FTIR、EDS對表面的元素官能基進行初步分析，並以XPS做為佐證，確認交聯的結構與穩定性；同時具備親水性與良好的生物相容性環境被生物醫學認為是細胞生長不可或缺的基本元素。最後將改質過後的基材，以L929上皮細胞進行繼代的細胞貼附性測試，結果顯示細胞的貼附性與生長的型態明顯較未改質之基材所培養出的優異，且與市售的細胞培養基材無明顯差異，但在後續的應用上卻遠多於目前市售的產品，如針對特定蛋白或加入胜肽做接枝；最後針對基材表面所交聯的薄膜中所含的物質進行單一化萃取，並以萃取液做體外細胞毒性測試（ISO 10993-5），了解薄膜中每種成分在環境中對細胞的影響，並找到最佳的培養條件。 The purpose of this study is to improve the surface biocompatibility of a polystyrene culture dish. The work used argon gas and oxygen in low-temperature vacuum plasma to generate polar groups such as C=O and -OH on the surface of the lipophilic culture dish substrate, thus the adhesion of other functional groups could be facilitated further to provide the surface with better cell affinity. Selecting microbial universal polyphenylene petri dish was as the substrate, the study first found the maximum tolerable plasma energy range and the surface activity degradation time for the substrate and then within the tolerable range, the optimal plasma exposure time, medium gas flow rate, wattage and other parameters for the best surface modification were achieved, and the corresponding surface properties were investigated.Initiator (Ammonium persulfate) and the inhibitor (Benzil α, α-dimethyl acetal) were added to the plasma-treated substrate for photo-grafting and crosslinking with acrylic acid molecules in an aqueous solution. A stable and hydrophilic film of poly (acrylic acid) was created on the substrate surface to provide a persistent surface activity over time. The film of poly(carboxylic acid) is considered to have a positive influence on the adhesion of growing cells, and the carboxyl groups of acrylic acid themselves can be used as an important key for subsequent modification; After grafting acrylic acid, this work uses EDC-NHS as an agent to graft amino acids onto the carboxylic groups of acrylic acid at a low temperature and in an acidic environment, to create a biologically beneficial growth environment. AFM was used to detect the roughness and mechanical properties of the surface, and the surface structure changes after modification were analyzed. The functional groups of the surface were analyzed by ATR-FTIR and EDS, and XPS was used as an evidence to confirm cross-linking and structural stability. Good hydrophilicity and biocompatibility are considered by biomedically essential for cell growth.Finally, the modified substrate was sub-cultured with L929 epithelial cells for cell adhesion test. The results showed that the cell adhesion and cell growth patterns were significantly better than the primitive substrate. In comparison, there is no significant difference between the commercial cell culture substrate and the product of this work. The treated substrates were soaked with solvent to extract the substance contained in the film on the substrate. The test for in vitro cytotoxicity (ISO 10993-5) was applied to investigate the effects of each component extracted on the culture environment and hence the best culture conditions could be found.
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