ST细胞无血清悬浮驯化及其在猪传染性胃肠炎疫苗中的应用

Serum-free suspension domestication of ST cells and its application in porcine transmissible gastroenteritis vaccine

为了将贴壁的ST细胞驯化为无血清悬浮生长的细胞用于猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)生物反应器规模化生产,试验采用逐步降低贴壁细胞培养基中血清含量的方法获得适应低血清生长的贴壁细胞,再将低血清生长的贴壁细胞驯化成无血清悬浮生长的ST细胞,并将其命名为ST-TY。在摇瓶试验中,按照MOI为0.01,0.05,0.1同步接入猪传染性胃肠炎病毒,分别在12,24,48小时时取样检测病毒效价。按照悬浮细胞的初始接种密度为0.6×10^(6)个/mL放大到15 L生物反应器,培养条件为pH值7.0~7.2、温度37℃、转速80~100 r/min、溶氧量40%~60%,培养24 h后收毒并检测病毒效价。结果表明:经无血清悬浮驯化后的ST-TY细胞增殖稳定,存活率在90%以上。ST-TY细胞接种猪传染性胃肠炎病毒的最佳条件为MOI=0.01,病毒最佳收获时间为24 h。在15 L生物反应器中,ST-TY细胞生长密度达到4.0×10^(6)个/mL,制备的猪传染性胃肠炎病毒效价为1×10^(7.80)TCID_(50)/mL,达到了标准要求。说明经无血清悬浮驯化的ST-TY细胞株增殖稳定,对猪传染性胃肠炎病毒敏感,可用于猪传染性胃肠炎病毒疫苗的生产。

In order to domesticate the adherent ST cells into serum-free suspension growth cells for the large-scale production of Transmissible gastroenteritis virus(TGEV)bioreactor,the experiment adopted the method of gradually reducing the serum content in the adherent cell culture medium to obtain adherent cells that were adapted to low serum growth,and then acclimated the adherent cells grown in low serum into serum-free suspension growth ST cells,named ST-TY.In the shake flask test,porcine Transmissible gastroenteritis virus was simultaneously inoculated with MOI of 0.01,0.05,and 0.1,and the samples were taken at 12,24,and 48 h to detect the virus titer.According to the initial seeding density of suspended cells of 0.6×10^(6) cells/mL,it was enlarged to a 15 L bioreactor,and the culture conditions were pH value 7.0-7.2,temperature for 37℃,rotation speed of 80-100 r/min,and dissolved oxygen content of 40%-60%.The virus was collected and the titer was detected after 24 h.The results showed that ST-TY cells proliferated stably after being acclimated by serum-free suspension,and the survival rate was over 90%.The best condition for ST-TY cell inoculation with porcine Transmissible gastroenteritis virus was MOI=0.01,and the best time for virus harvest was 24 h.In a 15 L bioreactor,the growth density of ST-TY cells reached 4×10^(6) cells/mL,and the titer of the prepared porcine Transmissible gastroenteritis virus was 1×10^(7.80) TCID_(50)/mL,which met the standard requirements.The results suggested that the ST-TY cell strain domesticated by serum-free suspension proliferated stably,which was sensitive to porcine Transmissible gastroenteritis virus,and can be used for the production of porcine transmissible gastroenteritis vaccine.