Optimization of lentivirus production process in a fixed-bed bioreactor
The lentivirus vectors can stably integrate exogenous genes into the genomes of various cells,making it useful in gene therapy.Among various bioreactors,fixed-bed bioreactors are increasingly used to scale-up the cultivation of adherent cells for the manufacture of lentivirus through plasmid transient transfection.In the fixed-bed bioreactor the production of lentivirus vectors can be heavily affected by various operating parameters.This study aims to optimize the transfection conditions and cell culture parameters during the production of lentivirus,and then validate optimized process conditions in China's domestically-made fixed-bed reactor.The results showed that during the plasmid transfection,the transfection efficiency could be significantly improved in the optimized conditions where the mass ratio of PEI to DNA was 2∶1,the DNA concentration during transfection was 2 μg/mL,and the transfection time was 6 hours,respectively.The mass ratio of PEI to DNA was the key factor to the success of transfection.When the ratio of PEI to DNA was below 2∶1,the transfection efficiency noticeably reduced and exogenous gene could not be expressed in the HEK293T cells.However,DNA concentration during mixing and mixing time have little effect on transfection efficiency,which mainly affects the expression of exogenous genes in cells.When the HEK293T cells were cultured in shaking bottles based on PET nonwoven fabric,the cells were evenly distributed on the surface of the scaffold and quickly entered the exponential growth phase at a high inoculation density of 5.0×104 cells/cm2.The cell densities that are too high or too low during transfection are not conducive to lentivirus production,and a high lentivirus titer can be obtained when the cell density is 1.0×106 cells/cm2.After process optimization,the lentivirus yield reached 2.4x1010 TU in the fixed-bed bioreactor with an surface of 2.0 m2.The results of this study can provide data support for the development and establishment of the production of lentivirus vectors based on a fixed-bed bioreactor.
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