Marc-145细胞微载体悬浮培养消化放大技术研究

Research on the scale-up technique of microcarrier suspension culture and digestion of Marc-145 cells

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.