生物反应器微载体放大培养Marc-145细胞及PRRSV增殖情况

Scale-up culture of Marc-145 cells by using microcarriers in various bioreactors and propagation of PRRSV

目的确定不同级别生物反应器间Marc-145细胞在微载体上消化放大培养条件,实现Marc-145细胞二级放大后,在生物反应器内增殖猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)。方法在一级生物反应器(BC-7L型)内,以微载体密度4 g/L培养Marc-145细胞,培养72 h时经灭菌PBS漂洗、胰酶消化后,接种至二级生物反应器(BC-14L型)继续培养,实现反应器间微载体细胞5倍体积增殖培养。以0.05 MOI接种PRRSV(TJM-F92株),接毒后24、36、48、60、72 h分别取上清及全液样品,检测病毒效价(TCID50)。结果 Marc-145细胞经过一级生物反应器培养72 h后,细胞密度达28.7×105个/ml;消化放大后,二级生物反应器培养72 h后,细胞密度达24.9×10~5个/ml。接毒后36 h,样品效价峰值可达108.19 TCID50,上清与全液样品效价差异较小。结论 Marc-145细胞从BC-7L型到BC-14L型不同反应器之间进行放大培养是可行的,为PRRSV活疫苗新型工艺改进及大规模放大生产奠定了基础。

Objective To investigate the condition for scale-up culture of Marc-145 cells by using microcarrires in various bioreactors and propagate porcine reproductive and respiratory syndrome virus （PRRSV）. Methods Marc145 cells were cultured in primary bioreactor （BC-7L） containing 4 g/L cytodex-1 for 72 h, then sterilized, rinsed, digested with trypsin and inoculated to secondary bioreactor （BC-14L） for propagation of the cells to 5 times of the original volume. PRRSV （TJM-F92 strain） was inoculated at a MOI of 0. 05, and the supernatant and suspension samples were collected 24, 36, 48, 60 and 72 h after inoculation and determined for virus titer （TCIDs0）. Results Marc-145 cells reached a density of 28. 7 × 105 cells / ml 72 h after culture in primary bioreactor, and 24. 9 × 105 cells / ml 72 h after culture in secondary bioreactor after digestion with trypsin. The virus titer reached a peak value of 108. 19 TCID50 36 h after inocu- lation, which showed no significant difference in supematant and suspension, Conclusion It was feasible to perform a scale-up culture of Marc-145 cells from BC-7L to BC-14L bioreactors, which laid a foundation of optimization of novel production procedure and large-scale production of live PRRSV vaccine.