文献类型：期刊文献

中文题名：微载体浓度与细胞接种密度对MDCK细胞生长的影响

英文题名：Effect of microcarrier concentration and cell density on growth of Madin-Darby canine kidney cells

作者：贾涵婧[1];王逸群[1];黄锭[1];罗剑[2];周琳婷[2];陈则[2];刘旭平[1];谭文松[1];周燕[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237;[2]上海生物制品研究所有限责任公司,上海200052

年份：2014

卷号：27

期号：9

起止页码：1138

中文期刊名：中国生物制品学杂志

外文期刊名：Chinese Journal of Biologicals

收录：CSTPCD;;Scopus;CSCD:【CSCD_E2013_2014】；

语种：中文

中文关键词：肾脏上皮细胞;犬;微载体;细胞密度

外文关键词：Kidney epithelium； Canine； Microcarrier； Cell density

摘要：目的探讨微载体浓度与犬肾脏上皮（Madin-Darby canine kidney,MDCK）细胞接种密度之间的关系,及其对MDCK细胞生长代谢的影响。方法实验设计了4组不同的单个微载体周围的细胞数范围,分别为0-10、10-20、20-60和60-120个,每组范围分别通过控制微载体浓度或细胞接种密度得到2组不同条件的实验组,即12 g/L微载体组和5×104个/ml MDCK细胞组、7 g/L微载体组和1×105个/ml MDCK细胞组、3 g/L微载体组和5×105个/ml MDCK细胞组、1 g/L微载体组和1×106个/ml MDCK细胞组,其中1、3、7、12 g/L微载体组细胞接种密度均为2×105个/ml,5×104、1×105、5×105、1×106个/ml MDCK细胞组微载体浓度均为3 g/L。经转瓶培养,每隔24 h取样,进行细胞计数,并检测细胞代谢。按确定的最佳细胞接种密度和微载体浓度培养MDCK细胞72 h后,接种0.01 MOI的甲型流感病毒株A/PR8/34,检测病毒的HA效价。结果当一个微载体周围的细胞数控制在20-60个之间,微载体浓度为3 g/L,细胞接种密度为5×105个/ml时,活细胞密度（viable cell density,VCD）可达3×106个/ml以上,细胞在72-96 h之间,能获得一个较为稳定的平台期,葡萄糖等营养物不会消耗过多以致耗竭,而乳酸、氨等代谢副产物的累积对培养环境造成的压力较小,有利于后期流感病毒的接种,同时还可达到理论最高活细胞值的60%以上。HA效价可达12 log2HA units/50μl。结论控制单个微载体周围细胞数在20-60个之间,细胞生长和代谢显著优于其他条件,且有利于病毒的复制,提高了病毒滴度。为基于MDCK细胞大规模高效生产流感病毒疫苗的工业化生产提供了数据参考。
Objective To investigate the relationship between microcarrier concentration and inoculum density of MadinDarby canine kidney（MDCK）cells as well as their effects on cell growth.Methods Four ranges of cell counts around a single microcarrier,0 - 10,10 - 20,20 - 60 and 60 - 120,were designed.To control the cell counts at the four designed ranges,the microcarrirer concentrations were designed as 12,7,3 and 1 g / L,while the inoculum densities of MDCK cells as 5 × 104,1 × 105,5 × 105 and 1 × 106 cells / ml,respectively.All the inoculum densities of MDCK cells for various microcarrier concentrations were 2 × 105 cells / ml,while all the microcarrirer concentrations for various inoculum densities were 3 g / L.The cells were culture in spinners,from which samples were taken every 24 h,counted and tested for metabolism.MDCK cells were inoculated at optimal density and cultured at optimal microcarrier concentration for 72 h,to which influenza virus A / PR8 / 34 strain was inoculated at a MOI of 0.01 and determined for HA titer.Results When the cell count around a microcarrier was controlled at 20 - 60,the microcarrier concentration was3 g / L,and the inoculum density of 5 × 105 cells / ml,the viable cell density（VCD）reached more than 3 × 106 cells / ml.The cell growth entered to a stable platform period after culture for 72 - 96 h,in which the nutrients such as glucosewere not consumed excessively or even exhausted,while the accumulation of metabolic by-products such as lactic acid and ammonia showed relatively low pressure to the culture environment.The condition was beneficial to the inoculation of influenza virus,under which the VCD may reach more than 60% of the theoretical maximum,and the HA titer may reach 12 log2 HA unites / 50 μl.Conclusion When the cell counts around a single microcarrier was controlled at 20 -60,both the cell growth and metabolism were superior to those under other conditions.The condition was beneficial to the virus replication and increase of virus titer,which provided a reference for large-scale production of MDCK cellsbased influenza vaccine.