Marc-145细胞微载体悬浮培养消化放大技术研究
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摘要
为了建立Marc-145细胞微载体培养放大技术,使其用于猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)疫苗的规模化生产,试验在7 L生物反应器内微载体培养Marc-145种子细胞,再用胰蛋白酶消化,然后进行逐级放大培养,并通过细胞最佳消化状态试验、保留胰蛋白酶消化液的影响试验、测试回收微载体试验等方法摸索胰蛋白酶消化放大技术参数。结果表明:Marc-145细胞在初级生物反应器内培养时,微载体使用量为5 g/L,细胞密度达3.18×10~6个/mL,并且在状态饱满时进行消化放大;在14 L生物反应器内培养时,Marc-145细胞均匀分布在微载体上并且代谢旺盛,在细胞密度为2.91×10~6个/mL时进行14 L消化放大;在14 L生物反应器内培养Marc-145细胞时,采用批培养方式,细胞比生长速率最大达0.031/h,在培养96小时时细胞密度达2.37×10~6个/mL。说明试验成功建立了生物反应器微载体培养Marc-145细胞的胰蛋白酶消化放大技术,并实现了逐级放大。
In order to establish the scale-up technique of microcarrier suspension culture of Marc-145 cells for the application of vaccine scale production of porcine reproductive and respiratory syndrome, a 7-liter bioreactor was used in this experiment for the microcarrier culture of seed Marc-145 cells,and then was digested with trypsin before the scale-up culture step by step. The scale-up technological parameters on trypsinization were explored through the methods including cell optimum digestion test,trypsin digestive liquid retaining effect test,microcarrier recovery measurement test. The results showed that when Marc-145 cells were cultured in the primary bioreactor,the amount of microcarriers was 5 g/L,and cell density was 3.18×10~6 cells/mL;then reaching full cell morphology,scale-up culture was carried out after digestion. When cultured in 14 L bioreactor,Marc-145 cells were uniformly distributed on microcarriers and were highly metabolized;when reaching a density of 2.91×10~6 cells/mL,the cells were scale-up inoculated to the tertiary bioreactor. When Marc-145 cells were cultured in 14 L bioreactor,batch culture mode was used and the specific cell growth rate reached the peak of 0.031/h;when 96 h post inoculation,cell density reached 2.37×10~6 cells/mL. The results indicated that the scale-up technique of microcarrier suspension culture and digestion of Marc-145 cells in the bioreactors was successfully established and the scale-up culture was achieved step by step.
In order to establish the scale-up technique of microcarrier suspension culture of Marc-145 cells for the application of vaccine scale production of porcine reproductive and respiratory syndrome, a 7-liter bioreactor was used in this experiment for the microcarrier culture of seed Marc-145 cells,and then was digested with trypsin before the scale-up culture step by step. The scale-up technological parameters on trypsinization were explored through the methods including cell optimum digestion test,trypsin digestive liquid retaining effect test,microcarrier recovery measurement test. The results showed that when Marc-145 cells were cultured in the primary bioreactor,the amount of microcarriers was 5 g/L,and cell density was 3.18×10~6 cells/mL;then reaching full cell morphology,scale-up culture was carried out after digestion. When cultured in 14 L bioreactor,Marc-145 cells were uniformly distributed on microcarriers and were highly metabolized;when reaching a density of 2.91×10~6 cells/mL,the cells were scale-up inoculated to the tertiary bioreactor. When Marc-145 cells were cultured in 14 L bioreactor,batch culture mode was used and the specific cell growth rate reached the peak of 0.031/h;when 96 h post inoculation,cell density reached 2.37×10~6 cells/mL. The results indicated that the scale-up technique of microcarrier suspension culture and digestion of Marc-145 cells in the bioreactors was successfully established and the scale-up culture was achieved step by step.
引文
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[2] 梅建国,王玉茂,王文秀,等.应用生物反应器生产高致病性猪繁殖与呼吸综合征病毒抗原[J].动物医学进展,2012,33(11):80-84.
[3] 过琴媛,王辉,沈心亮.微载体培养动物细胞技术的研究进展[J].微生物学免疫学进展,2007,35(1):73-75.
[4] LAM A T,CHEN A K,LI J,et al.Conjoint propagation and differentiation of human embryonic stem cells to cardiomyocytes in a defind microcarrier culture [J].Stem Cell Res Ther,2014,5(5):110.
[5] KIM B J,ZHAO T,YOUNG L,et al.Batch,fed-batch,and microcarrier cultures with CHO cell lines in a pressure-cycle driven miniaturized bioreactor[J].Biotechnol Bioeng,2012,109(1):137-145.
[6] 徐家华,陈瑞爱,唐秀英,等.动物细胞规模化培养及其在兽用疫苗生产中的应用[J].动物医学进展,2009,30(5):105-108.
[7] 冯见.一种新的Vero细胞微载体放大培养工艺研究[D].上海:上海交通大学,2012.
[8] LIU C C,LIAN W C,BUTLER M,et al.High immunogenic enterovius 71 strain and its production using serum-free microcarrier Vero cell culture[J].Vaccine,2007,25(1):19-24.
[9] AYYILDIZ-TAMIS D,AVCI K,DELILOGLU-GURHAN S I.Comparative investigation of the use of various commercial microcarriers as a substrate for culturing mammalian cells[J].In Vitro Cell Dev Biol Anim,2014,50(3):221-231.
[10] BAI R,GAO G,XING Y,et al.Two outward potassium current types are expressed during the neural differentiation of neural stem cells[J].Neural Regen Res,2013,8(28):2656-2665.
[11] 盛程程,冯二凯,任飞,等.Vero细胞生物反应器微载体悬浮培养工艺的建立[J].黑龙江畜牧兽医,2018(01):186-189,257-258.
[12] BOCK A,SANN H,SCHULZE-HORSEL J,et al.Growth behavior of number distributed adherent MDCK cells for optimization in microcarrier cultures[J].Biotechnol Prog,2009,25(6):1717-1731.