聚N-异丙基丙烯酰胺改性聚苯乙烯培养皿:以温度变化控制细胞的分离

Poly N-isopropylacrylamide grafted tissue culture polystyrene dish for controlling cell detachment based on temperature changes

背景:在组织工程研究中,最常用的细胞分离方法是胰蛋白酶消化,但是这种方法可能破环细胞外基质蛋白造成细胞活性降低,因此研制一种避免酶消化的细胞分离技术将有重要的意义。目的:利用聚N-异丙基丙烯酰胺能够随温度变化疏水性的特性,建立一种基于温度敏感性控制细胞分离技术。方法:配制质量浓度为55%的异丙基丙烯酰胺异丙醇溶液,均匀涂抹在聚苯乙烯培养皿表面,然后通过电子束照射的方法接枝于培养皿表面。用原子力显微镜观察接枝前后聚苯乙烯培养皿表面微观结构,静滴法测量不同温度下接枝前后聚苯乙烯培养皿表面的水接触角,聚N-异丙基丙烯酰胺接枝后培养皿在不同温度下细胞分离能力变化。结果与结论:原子力显微镜观察发现聚苯乙烯培养皿表面上形成薄层致密的聚N-异丙基丙烯酰胺聚合物;在37℃时聚N-异丙基丙烯酰胺接枝培养皿与未接枝培养皿水接触角差异无显著性意义,在20℃时聚N-异丙基丙烯酰胺接枝培养皿的水接触角明显低于未接枝培养皿;在37℃时细胞能正常的在温度敏感性培养皿上贴壁生长,在20℃时大量细胞从培养皿底面脱离悬浮在培养基中。结果说明聚N-异丙基丙烯酰胺接枝培养皿表面可以利用温度的改变控制细胞的分离,此技术有望在组织工程中发挥重要的作用。

BACKGROUND：In the tissue engineering studies,the common method of cell detachment is trypsinization.However,this method may decrease cell activity by breaking the extracellular matrix proteins.Therefore,it is important to develop a new cell detachment method which can avoid the damage from trypsinization.OBJECTIVE：Based on that poly N-isopropylacrylamide（PNIPAAm）could alter its hydrophobic property with the change of temperature,this study aimed to develop a novel method which can control cell detachment by simply changing the temperature.METHODS：IPAAm was dissolved in isopropanol with final concentration of 55%（w/v）.IPAAm solution was evenly laid on the surface of TCPS dish,and PNIPAAm was then covalently grafted on the TCPS dish surface by electron beam irradiation.The microstructure of the surface of TCPS dish was observed by atomic force microscopy（AFM）before and after PNIPAAm grafted.Water contact angle was determined by sessile drop method.And an inverted phase contrast microscope was used to observe and evaluate the cell detachment ability of PNIPAAm grafted TCPS dish by subjecting the same dish to different temperatures.RESULTS AND CONCLUSION：AFM images showed that a thin and compact PNIPAAm layer formed on the TCPS dish surface.In the water contact angle test,there was no statistic difference between PNIPAAm grafted and ungrafted surfaces at 37℃,and the water contact angle of PNIPAAm grafted surfaces was lower than that of ungrafted surface at 20℃.The cells adhered to the surface of the PNIPAAm grafted TCPS dish at 37℃,which would be detached at 20℃.It was successful to control cell detachment by simply changing the temperature with PNIPAAm grafted TCPS dish,and it would be promising to be applied in tissue engineering.