探討溫度響應高分子複合薄膜應用於細胞貼附與細胞團簇

Investigate the temperature-responsive polymer composite film for cell attachment and spheroid formation

楊凱文(KAI-WEN YANG)

指導教授 ： 葛宗融

中原大學/理學院/奈米科技碩士學位學程/碩士(2022年)

https://doi.org/10.6840/cycu202301062

摘要

本研究主要以靜電紡絲技術製備一複合纖維膜做為細胞培養基材，並利用材料的溫度響應特性將培養於其上的細胞進行分離。將聚N-異丙基丙烯醯胺的溫度響應特性和聚己內酯的可塑性及生物相容性結合做為紡絲溶液的核心材料，製備出具溫度響應特性、高比表面積、高孔洞性的紡絲纖維結構。
本研究對不同比例的複合纖維膜進行材料驗證並應用於細胞培養，在場發射電子顯微鏡的結果顯示直徑粗度皆在0.4 ~1.8 µm。紅外光光譜分析儀及接觸角量測驗證了其在不同組成下的表面官能基成分及親疏水性的變化，在機械拉伸試驗的部分，觀察到聚己內酯增加了此複合紡絲薄膜的可承受形變，在溫度調控的溶液下驗證面積收縮率，結果顯示比例為4:1的複合紡絲纖維薄膜有55%的面積收縮。運用L929小鼠纖維母細胞共培養進行毒性測試後的細胞存活率皆高於70%，並嘗試細胞分離後觀察到細胞團簇的行為。於培養4T1乳腺癌細胞時，歸納了不同生長時間下去貼附所產生的團簇大小，團簇直徑落在100 µm至500 µm，經計算後小型團簇的平均面積為3530.22±1408.29 µm^2；大型團簇平均面積為14946.38±3557.02 µm^2。
本研究成功利用此複合紡絲纖維薄膜的溫度響應特性將細胞分離於此細胞培養平台，並期望未來可進一步將此複合薄膜應用於培養不同腫瘤細胞株亦或是在腫瘤生長的微環境上有所應用。

關鍵字

靜電紡絲 ； 溫度響應高分子 ； 細胞貼附 ； 細胞團簇

並列摘要

In this study, a composite fiber membrane was prepared by electrospinning technology as a cell culture substrate. Cultured cells on it, separated by the temperature response characteristics of the material. The temperature response characteristics of poly(N-isopisopropyl acrylamide)(PNIPAAm) and the elasticity, and biocompatibility of polycaprolactone (PCL) were used as the core material of the spinning solution, to prepare a membrane with temperature-responsive properties, high specific surface area, and high porosity.
In this study, the composite fiber membranes with different ratios were verified and applied to cell culture. The results of field emission Scanning Electron Microscope (SEM) showed that the diameter ranged from 0.4 to 1.8 μm. The results of infrared spectroscopy (FTIR) and contact angle measurement verified the surface functional groups compositions and hydrophilic-hydrophobic properties of different ratio compositions. In the part of the mechanical tensile test, it was observed that polycaprolactone improved the withstand deformation of the composite film. The area shrinkage rate was verified by sinking in the temperature-controlled solution, and showed that the composite spun fiber film with a ratio of 4:1 had a 55% area shrinkage. After that, it was put into co-culture with L929 mouse fibroblasts. The cell viability was higher than 70 % after the toxicity test, and the behavior of cell clusters was observed after detaching cells. When applied to co-culture with 4T1 breast cancer cells, the size of the clusters generated by detaching in different growth stages was observed and summarized. The diameter of the clusters was from 100 to 500 µm, the average area of small clusters was 3530.22±1408.29 µm2, and the average area of large clusters was 14946.38±3557.02 µm2.
In this study, the temperature-responsive properties of the composite spun fiber film was successfully used to separate cells from this cell culture membrane, and it is expected that the composite film can be further applied to culture the different tumor cell lines in the future, or have the potential to investigate the microenvironment of tumor growth.

並列關鍵字

electrospinning ； temperature-sensitive polymers ； cell attachment ； cell clusters

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