Studies On Suspension Cell Line Establishment And Culture Of Marc-145

Porcine reproductive and respiratory syndrome virus is an infectious disease,which can cause reproductive failure in sows and respiratory symptoms in piglets.It has high infectivity and mortality throughout almost all the pig countries,causing serious economic losses to the pig industry and vaccination remains the most effective measure.The production technology of adherent culture of Marc-145 proliferating virus by rotating bottle are adopted by producing PRRS vaccination at home and abroad.The production of vaccine by bioreactor suspension culture cells is the only way to transform and upgrade for bio pharmaceutical industry in the world.This study choose the method of reducing serum sharply and gradually,and then a suspension cell was obtained by using silicified system of shaking flask-table to suspension culture adherent Marc-145 cells in low serum medium after 22 generations continuously.Its characteristics of large scale culture and the ability of virus multiplication in 5 L bioreactor were also studied.The main results are as follows:1.An adherent Marc-145 cell strain was introduced from CCTCC,then amplification culture to establish the cell bank with 40 frozen cell strains and to evaluate their biological characteristics.The results of sterility test,mycoplasma examination,adventitious agent were all negative.The growth of recovered Marc-145 cells was stable,the growth curve was closely typical and basically "S" type,the average activity reached 97%.the maximum proliferation density was 57.8×104 cells/mL,the doubling time was 30.2 hours.2.The serum levels of adherent Marc-145 cells were decreased from 10%to 2%by using the method of reducing serum sharply and gradually,and an adherent Marc-145 cell strain with 2%serum was obtained.Then it was passaged by 1:3,which grew steadily,the morphology was good,the maximum proliferation density of cells was 40.3×104 cells/mL,the cell viability was about 98.5%,and the population doubling time was 51.9 h.3.A suspension cell was obtained by using silicified system of shaking flask-table to suspension culture adherent Marc-145 cells in low serum medium after 22 generations continuously,which was named Marc-145-XF.Then amplification culture Marc-145-XF to establish the cell bank.The results of sterility test,mycoplasma examination,adventitious agent were all negative.And the result of chromosome examination was agree with Marc-145.After culturing 8 generations,the cells grew normally and the morphology was uniform.It would be best that to subculture Marc-145-XF cells with 0.5×106 cells/mL in 2%newborn bovine serum medium for 48 h,the maximum proliferation density was 3.22×106 cells/mL,the doubling time was 27 h.4.The virus titer of Marc-145-XF cells was TCIDso=10-4.74/0.1ml,which was inoculated with porcine blue ear virus.5.When using the 5L bioreactor for large-scale culture Marc-145 cells,the culture temperature was 36.5 ? the speed was 70 r/min,the pH maintained at 6.5-7.15,the dissolved oxygen was 40%;it reached the maximum proliferation density of?2.85 x 106 cells/mL after culturing 96 h.