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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 determine the digestive and enveloping conditions of Marc-145 cells in different bioreactors on microcarriers. After two-step amplification of Marc-145 cells, porcine reproductive and respiratory syndrome virus was proliferated in the bioreactor virus, PRRSV). Methods Marc-145 cells were cultured in primary bioreactor (BC-7L) with microcarrier density of 4 g / L. After culturing for 72 h, they were rinsed with sterile PBS, digested with trypsin and inoculated into secondary bioreactor (BC-14L type) continue to train to achieve a 5-volume proliferation of microcarrier cells between the reactor proliferation. PRRSV (TJM-F92 strain) was inoculated with 0.05 MOI. The supernatant and whole blood samples were taken at 24, 36, 48, 60 and 72 h after receiving the virus and the virus titer (TCID50) was determined. Results The cell density of Marc-145 cells reached 28.7 × 105 cells / ml after being cultured for 72 hours in a bioreactor. After digestion and amplification, the cell density reached 24.9 × 10-5 cells / ml. 36 h after receiving the drug, the peak value of the sample reached 108.19 TCID50, the difference between the supernatant and the whole liquid sample was small. Conclusions It is feasible to amplify Marc-145 cells from BC-7L to different reactors of BC-14L and lay a foundation for new process improvement and large-scale production of live vaccine of PRRSV.