转瓶培养与生物反应器微载体培养重组腺病毒的比较
|
摘要
目的分别用3L转瓶和7L生物反应器微载体系统培养人胚胎肾细胞(HEK-293细胞)并接种H3型流感重组腺病毒,比较两种方法培养细胞及重组腺病毒的效果。方法分别用3L转瓶和7L生物反应器培养HEK-293细胞。转瓶每24h取样,生物反应器每12h取样,记录细胞总数。待细胞长满表面85%~90%时接种流感重组腺病毒,完全病变后收取病毒液并测定病毒滴度。结果转瓶培养HEK-293细胞3d,细胞增殖增加1.85~2.45倍,为(3.71~4.90)×107个[(2.95~3.90)×104 cells/cm2],生物反应器培养3d,细胞增长4.72~5.00倍,为(132.15~140.09)×107个[(24.88~28.72)×104cells/cm2]。转瓶培养重组腺病毒滴度为6.85TCID50/ml,生物反应器微载体系统培养腺病毒滴度为7.200TCID50/ml。结论生物反应器微载体系统培养流感重组腺病毒数量和滴度均高于转瓶培养,不但提高了疫苗的产量,还减少了细胞分泌物的残留,增强了疫苗接种的安全性。
Objectives To use 3-L roller bottles and a 7-L bioreactor system to culture a recombinant adenovirus containing H3 influenza in human embryonic kidney cells(HEK-293)and to compare the effectiveness of the two methods of culturing cells and a recombinant adenovirus. Methods HEK-293 cells were cultured in 3-L roller bottles and a 7-L bioreactor system.Inoculation density was 1.6×104 cells/cm2.The total number of cells in a roller bottle was recorded every 24 hours and the number in the bioreactor was recorded every 12 hours.When cells covered 85%-90% of the available surface,cells were inoculated with the recombinant adenovirus and the viral titer was determined. Results After 3days of culturing HEK-293 cells in roller bottles,cell growth increased 1.85-2.45-fold,ultimately reaching 3.71-4.90×107cells(2.95-3.90×104 cells/cm2).After 9days,cells covered the entire wall of the bottle,and the total cell count reached 31.25-36.07×107cells(24.88-28.72×104 cells/cm2).The cell count increased 4-fold.The growth of HEK-293 cells in the bioreactor system increased 4.72-5.00-fold and the total cell count increased to 132.15-140.09×107cells(24.88-28.72×104 cells/cm2).The titer of the recombinant adenovirus was 6.85TCID50/ml in roller bottles and7.20TCID50/ml in the bioreactor system. Conclusion After 3days,the density of HEK-293 cells cultured in the bioreactor system was 29 times that in roller bottles.The proliferation of adenovirus in the bioreactor system was 6.4times that in roller bottles.The recombinant adenovirus cell count and the titer were higher in the bioreactor system.The approach used here could increase production of a vaccine,reduce cell secretions,and increase the safety of vaccination.
Objectives To use 3-L roller bottles and a 7-L bioreactor system to culture a recombinant adenovirus containing H3 influenza in human embryonic kidney cells(HEK-293)and to compare the effectiveness of the two methods of culturing cells and a recombinant adenovirus. Methods HEK-293 cells were cultured in 3-L roller bottles and a 7-L bioreactor system.Inoculation density was 1.6×104 cells/cm2.The total number of cells in a roller bottle was recorded every 24 hours and the number in the bioreactor was recorded every 12 hours.When cells covered 85%-90% of the available surface,cells were inoculated with the recombinant adenovirus and the viral titer was determined. Results After 3days of culturing HEK-293 cells in roller bottles,cell growth increased 1.85-2.45-fold,ultimately reaching 3.71-4.90×107cells(2.95-3.90×104 cells/cm2).After 9days,cells covered the entire wall of the bottle,and the total cell count reached 31.25-36.07×107cells(24.88-28.72×104 cells/cm2).The cell count increased 4-fold.The growth of HEK-293 cells in the bioreactor system increased 4.72-5.00-fold and the total cell count increased to 132.15-140.09×107cells(24.88-28.72×104 cells/cm2).The titer of the recombinant adenovirus was 6.85TCID50/ml in roller bottles and7.20TCID50/ml in the bioreactor system. Conclusion After 3days,the density of HEK-293 cells cultured in the bioreactor system was 29 times that in roller bottles.The proliferation of adenovirus in the bioreactor system was 6.4times that in roller bottles.The recombinant adenovirus cell count and the titer were higher in the bioreactor system.The approach used here could increase production of a vaccine,reduce cell secretions,and increase the safety of vaccination.
引文
[1]Easterday BC,Hinshaw VS,Halvorson DA,et al.Influenza:Disease of poultry[M].Iowa State University Press,Ames:IA,USA,1997:583-605.
[2]解林红,高晓龙,耿海东,等.H4N6亚型禽流感病毒分离与氨基酸序列测定[J].中国病原生物学杂志,2015,10(5):385-8.
[3]孙新城,王云龙,李玉林,等.甲型流感H1N1(2009)流感病毒M2e单克隆抗体的制备及双抗体夹心ELISA检测方法的建立[J].中国病原生物学杂志,2015,10(6):508-11,524.
[4]Watanabe T,Kawaoka Y.Pathogenesis of the 1918pandemic influenza virus[J/OL].Plos Pathog,2011,7(1):e1001218.
[5]Laver G,Garman E.Virology-The origin and control of pandemic influenza[J].Science,2001,293(5536):1776-7.
[6]王铁成,任志广,桑晓宇,等.A型流感病毒通用型人源化抗体的构建及初步鉴定[J].中国病原生物学杂志,2014,9(6):481-5,560.
[7]靖杰,鲁会军,刘昊,等.Quil A为佐剂的H1、H3亚型流感病毒多表位DNA疫苗免疫评价[J].中国病原生物学杂志,2013,8(1):1-4.
[8]国家食品药品监督管理局.人基因治疗研究和制剂质量控制技术指导原则[EB/OL].[2003-03-20](2015-09-12).http://www.sda.gov.cn/WS01/CL0237/15708.html.
[9]Trapnell BC.Adenoviral Vector for Gene Transfer[J].Adv Drug Deliv Rev,1993(12):185-99.
[10]Van Wezel A.Growth of cell-strains and primary cells on microcarriers in homogeneous culture[J].Nature,1967,21(6):64-5.
[11]Tree JA,Richardson C,Fooks AR,et al.Comparison of largescale mammalian cell culture systems with egg culture for the production of influenza virus A vaccine strains[J].Vaccine,2001,19(25-26):3444-50.
[12]No authors.Requirement for use of animal cells as in vitro substrates for the production of biologicals[J].WHO Technical Reptor Series 1998(878):19-56.
[13]Jayakumar R,Venkatesh G,Albert H,et al.Vero cell rabies vaccine for animals in miceocarrier culture[J].Indian J Anim Sci,2002,72(7):549-50.
[14]Ehrlich HJ,Muller M,Oh HM,et al.Aclinical trial of a wholevirus H5N1vaccine derived from cell culture[J].N Engl J Med,2008,358(24):2573-84.
[15]Healthcare GE.Microcarrier cell culture principles and methods[M].Uppsala:Pharmacia Fine Chemicals AB,1981.
[16]Klyushnichenko V,Bernier A,Kamen A,et al.Improved highperformance liquid chromatographic method in the analysis of adenovirus particles[J].J Chromatogr B,2001(755):27-36.
[17]Transfiguracion J,Bernier A,Arcand N,et al.Validation of a high-performance liquid chromatography assay for the quantification of adenovirus type 5particles[J].J Chromatogr B,2001(761):187-94.
[18]Nadeau I,Kamen A.Production of adenovirus vector for gene therapy[J].Biotech Adv,2003(20):475-89.
[2]解林红,高晓龙,耿海东,等.H4N6亚型禽流感病毒分离与氨基酸序列测定[J].中国病原生物学杂志,2015,10(5):385-8.
[3]孙新城,王云龙,李玉林,等.甲型流感H1N1(2009)流感病毒M2e单克隆抗体的制备及双抗体夹心ELISA检测方法的建立[J].中国病原生物学杂志,2015,10(6):508-11,524.
[4]Watanabe T,Kawaoka Y.Pathogenesis of the 1918pandemic influenza virus[J/OL].Plos Pathog,2011,7(1):e1001218.
[5]Laver G,Garman E.Virology-The origin and control of pandemic influenza[J].Science,2001,293(5536):1776-7.
[6]王铁成,任志广,桑晓宇,等.A型流感病毒通用型人源化抗体的构建及初步鉴定[J].中国病原生物学杂志,2014,9(6):481-5,560.
[7]靖杰,鲁会军,刘昊,等.Quil A为佐剂的H1、H3亚型流感病毒多表位DNA疫苗免疫评价[J].中国病原生物学杂志,2013,8(1):1-4.
[8]国家食品药品监督管理局.人基因治疗研究和制剂质量控制技术指导原则[EB/OL].[2003-03-20](2015-09-12).http://www.sda.gov.cn/WS01/CL0237/15708.html.
[9]Trapnell BC.Adenoviral Vector for Gene Transfer[J].Adv Drug Deliv Rev,1993(12):185-99.
[10]Van Wezel A.Growth of cell-strains and primary cells on microcarriers in homogeneous culture[J].Nature,1967,21(6):64-5.
[11]Tree JA,Richardson C,Fooks AR,et al.Comparison of largescale mammalian cell culture systems with egg culture for the production of influenza virus A vaccine strains[J].Vaccine,2001,19(25-26):3444-50.
[12]No authors.Requirement for use of animal cells as in vitro substrates for the production of biologicals[J].WHO Technical Reptor Series 1998(878):19-56.
[13]Jayakumar R,Venkatesh G,Albert H,et al.Vero cell rabies vaccine for animals in miceocarrier culture[J].Indian J Anim Sci,2002,72(7):549-50.
[14]Ehrlich HJ,Muller M,Oh HM,et al.Aclinical trial of a wholevirus H5N1vaccine derived from cell culture[J].N Engl J Med,2008,358(24):2573-84.
[15]Healthcare GE.Microcarrier cell culture principles and methods[M].Uppsala:Pharmacia Fine Chemicals AB,1981.
[16]Klyushnichenko V,Bernier A,Kamen A,et al.Improved highperformance liquid chromatographic method in the analysis of adenovirus particles[J].J Chromatogr B,2001(755):27-36.
[17]Transfiguracion J,Bernier A,Arcand N,et al.Validation of a high-performance liquid chromatography assay for the quantification of adenovirus type 5particles[J].J Chromatogr B,2001(761):187-94.
[18]Nadeau I,Kamen A.Production of adenovirus vector for gene therapy[J].Biotech Adv,2003(20):475-89.