无血清全悬浮培养Vero细胞系的建立及生物反应器培养参数优化  

Establishment of Vero Cell Line in Serum-free Suspension Culture and Optimization of Bioreactor Culture Parameters

作　　者：杨惠清 武发菊 葛玉凤 刘小刚 安芳兰 刘学荣 胡永浩[1] YANG Hui-qing;WU Fa-ju;GE Yu-feng;LIU Xiao-gang;AN Fang-lan;LIU Xue-rong;HU Yong-hao(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou,Gansu,730000,China;China Agricultural Vet Bio Science and technology CO.LTD,Lanzhou,Gansu,730046,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730000 [2]中农威特生物科技股份有限公司,甘肃兰州730046

出　　处：《动物医学进展》2023年第1期54-59,共6页Progress In Veterinary Medicine

基　　金：甘肃省成果转化资助专项(17ZD4CA010)。

摘　　要：为建立无血清全悬浮培养Vero细胞系,并使其在反应器中高密度生长。从美国典型培养物保藏中心(ATCC)引进贴壁培养型Vero细胞,通过降低血清浓度和改变培养液的方式获得1株无血清全悬浮培养型Vero细胞,扩增培养后液氮冻存,建立相应的细胞库;复苏细胞后对活力、形态、生长曲线、微生物污染、染色体核型等特性进行检定;通过Box-Behnken试验对生物反应器pH、溶氧和搅拌转速等3个培养参数进行优化,确定最优培养参数。结果显示,筛选的Vero悬浮细胞形态呈圆形、饱满、透亮,大小均匀,呈现较好的悬浮生长状态;生长曲线呈S型;细菌、真菌、支原体及外源病毒检查均为阴性;染色体数目为58或60的占75%;用响应面分析法得出的最优培养参数培养Vero细胞,细胞最高密度可达4.5×10^(6)cells/mL。结果表明,成功筛选出了适应无血清悬浮培养的Vero细胞,通过优化培养参数,可在生物反应器中高密度生长。In order to establish serum-free suspension culture Vero cell line and make it grow in high density in the reactor,the adherent culture Vero cell line was introduced from ATCC,and the suspension culture was acclimated by lowering serum and changing culture medium.The suspension culture cells obtained by acclimation were subcultured,amplified,and cryo-stored with liquid nitrogen to establish the corresponding cell bank.Observations on vigor,morphology,dynamic growth,microbial contamination and analysis of karyotype were carried out.The pH,dissolved oxygen and agitator shaft speed of the bioreactor were optimized by Box-Behnken test,and the optimal culture parameters were determined.The results showed that the suspended Vero cells were round,full and bright,with uniform size and showing agood suspended growth state.The growth curves of Vero cell lines were all S style.The tests for bacteria,fungi,mycoplasma and virus were all negative.The proportion of cells with 58 or 60chromosomes was 75%.The maximum density of the suspension cell line could increase to 4.5×10^(6) cells/mL by using theoptimal culture parameters obtained by response surface methodology.The results showed that Vero cells adapted to serum-free suspension culture were successfully screened out and could grow in high density in the bioreactor by optimizing the culture parameters of the bioreactor.

关 键 词：VERO细胞 细胞驯化 细胞库 生物反应器 

分 类 号：S852.163[农业科学—基础兽医学]