伍胤杰, 张名, 周健, 周玮, 杨希, 蒋润佳, 孙明波. 微载体预处理方式对其灭菌温度及Vero细胞贴壁率的影响[J]. 云南大学学报(自然科学版), 2017, 39(5): 876-882. doi: 10.7540/j.ynu.20170133
引用本文: 伍胤杰, 张名, 周健, 周玮, 杨希, 蒋润佳, 孙明波. 微载体预处理方式对其灭菌温度及Vero细胞贴壁率的影响[J]. 云南大学学报(自然科学版), 2017, 39(5): 876-882. doi: 10.7540/j.ynu.20170133
WU Yin-jie, ZHANG Ming, ZHOU Jian, ZHOU Wei, YANG Xi, JIANG Run-jia, SUN Ming-bo. Effects of microcarrier pretreatment methods on sterilization temperature and adherence rate of Vero cells[J]. Journal of Yunnan University: Natural Sciences Edition, 2017, 39(5): 876-882. DOI: 10.7540/j.ynu.20170133
Citation: WU Yin-jie, ZHANG Ming, ZHOU Jian, ZHOU Wei, YANG Xi, JIANG Run-jia, SUN Ming-bo. Effects of microcarrier pretreatment methods on sterilization temperature and adherence rate of Vero cells[J]. Journal of Yunnan University: Natural Sciences Edition, 2017, 39(5): 876-882. DOI: 10.7540/j.ynu.20170133

微载体预处理方式对其灭菌温度及Vero细胞贴壁率的影响

Effects of microcarrier pretreatment methods on sterilization temperature and adherence rate of Vero cells

  • 摘要: 目前,商品化的微载体多以干粉状态储存和销售,在细胞培养前需要先进行预处理,即用无Ca2+、Mg2+的磷酸盐缓冲液(phosphate-buffered saline,PBS)溶液对干粉微载体进行水合吸胀和洗涤,然后将水合吸胀后的微载体进行高压蒸汽灭菌处理,之后才能够用于细胞的培养.在这个过程中,可能会对微载体产生影响,从而影响细胞的贴壁和生长.作者将Cytodex1微载体用无Ca2+、Mg2+的PBS以50、25、16.7g·L-1 3种水合吸胀质量浓度,每种浓度以720、2400、6000mL 3种装量进行预处理.然后置于高压灭菌器中进行121℃高压蒸汽灭菌,观察其水合吸胀浓度及装量差异对灭菌温度造成的影响.灭菌完成后取样对微载体进行显微镜观察,然后将各组微载体样品用于Vero细胞的7L生物反应器培养,12h后记录各组的贴壁率.实验证明,对微载体进行预处理时,不同水合吸胀质量浓度和装量会对其灭菌时的升温速率造成影响.预处理时水合吸胀质量浓度不超过50g·L-1,121℃以上灭菌时间不超过212min,对Cytodex1微载体物理特性和Vero细胞的贴壁率没有影响(P>0.05).同时,在利用生物反应器培养Vero细胞时,除了考虑微载体的水合吸胀浓度和灭菌温度差异对细胞贴壁率的影响外,更重要的是预处理时不同水合吸胀浓度和装量所造成的灭菌温度差异是否能够满足无菌控制的要求.

     

    Abstract: At present,commercial microcarriers are stored and soled as dry powder.The powder is pretreated before cell culture,which is hydrated,imbibed and washed with PBS(no Ca2+,Mg2+),then is sterilized by steam.But during the progress,microcarrier could be affected and then influence the adherence rate of Vero cell. In this study,the Cytodex1 was pretreated by PBS(no Ca2+,Mg2+) at three concentration:1∶20(50g·L-1),1∶40(25g·L-1),1∶60(16.7g·L-1),each concentration with three different loading as 720,2400mL and 6000mL,and then steam sterilized at 121℃,to observe the effect of sterilization temperature caused by different concentration and loading.After the sterilization,the samples were observed under microscope,the microcarrier samples were used for the 7 L bioreactor culture of Vero cells.After 12 h,the adherence rate of each group was recorded.The results showed that the heating rate was affected by the concentration and loading amount when the carrier was pretreated.When the concentration was less than 50g·L-1,the sterilization was less than 212min above 121 ℃,the Cytodex1’s physical characteristics and Vero cell adhesion rate were not affected (P>0.05).During the culture of Vero cells,in addition to the adherent rate,whether the sterile control could meet the requirement under the different sterilization temperatures caused by different pretreatment conditions is more important.

     

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