摘要
目的采用96孔平底培养板结合琼脂糖凝胶建立人肝癌HepG2细胞三维培养模型,并选择适合的细胞活力检测方法。方法预处理96孔板,每孔加入1%琼脂糖溶液冷却后备用。将HepG2细胞以1.975×10~4、2.96×10~4、4.444×10~4、6.667×10~4、1×10~5个·mL^(-1)每孔100 μL接种于预处理的96孔板,通过倒置的显微镜观察细胞球形态并计算细胞球体积。利用酸性磷酸酶法(APH)和噻唑蓝法(MTT)测定细胞活力,比较两种方法的优劣。结果 HepG2细胞在每孔的接种量为1.975×10~3~1×10~4个时,在琼脂糖凝胶表面形成较为规则的细胞球,随着细胞接种数量的增加,细胞球的体积逐渐增大,在每孔1.975×10~3~6.667×10~3个时细胞呈现出较好的线性关系。APH法及细胞球经消化处理后的MTT法检测结果均显示,随着细胞接种数量的增加,吸光度逐渐增加,呈现出较好的线性关系,但细胞球消化操作复杂。结论采用96孔平底培养板结合琼脂糖凝胶培养法建立人肝癌HepG2细胞三维培养模型,并结合APH法进行细胞活力检测,此方法简单、便捷、准确、经济,可用于进一步的药物活性筛选及机制研究。
Objective To establish a 3-dimensional culture model of HepG2 hepatoma cells by a 96-well cell culture plate(flat bottom)and agarose,and to select a suitable cell viability detection method.Methods The 96-well plate pretreatment:1%agarose solution was added in each well to wait for the solidification.HepG2 cells were prepared 100μL per well at 1.975×104,2.96×104,4.444×104,6.667×104,and 1×105·mL-1 respectively.The shapes of cell spheroids were observed by inverted microscope and the volumes of cell spheroids were calculated.Acid phosphatase method(APH)and thiazolyl blue tetrazolium bromide method(MTT)were used to determine the cell viability,and the advantages and disadvantages of different methods were compared.Results HepG2 cells were cultured on the surface of agarose gel at 1.975×103-1×104 each well,to regular cell spheroids.As the number of inoculated cells increased,the volume of cell spheroids increased gradually,showing a good linear relationship at 1.975×103-6.667×103.APH method showed that with the increase in the number of inoculated cells,the absorbance gradually increased,showing a good linear relationship.MTT method showed that after the digestion,the absorbance gradually increased with the increase in the number of inoculated cells,showing a good linear relationship,but the digestion operation of cell spheroids was complex.Conclusion Three-dimensional culture model of HepG2 hepatoma cells is established by a 96-well cell culture plate(flat bottom)and agarose culture.The cell viability is detected by APH method.This method is simple,convenient,accurate and economical,and can be used for further drug screening and mechanism research.
作者
张静
樊雪
李淼
窦明杰
汪海峰
ZHANG Jing;FAN Xue;LI Miao;DOU Ming-jie;WANG Hai-feng(College of Pharmaceutical Sciences and Bioengineering,Shenyang University of Chemical Technology,Shenyang 110142;Liaoning Cheng Da Biological Co.Ltd,Shenyang 110179)
出处
《中南药学》
CAS
2020年第7期1111-1114,共4页
Central South Pharmacy
基金
2019年辽宁省大学生创新创业训练计划资助项目(No.201910149156)。
