Abstract: 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 2.4×1010 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.

Key words: fixed-bed bioreactor, lentivirus, transfection conditions, cell culture parameters

摘要： 慢病毒载体能将外源基因稳定整合至多种细胞的基因组，在基因治疗领域中具有广泛应用。固定床反应器可规模化培养贴壁细胞并通过质粒瞬时转染的方式生产慢病毒，但其慢病毒产量受到各种操作参数的影响。本研究对慢病毒生产过程中质粒转染条件和细胞培养参数进行优化，并在国产固定床反应器中验证了优化后的工艺。结果表明，在质粒转染过程中，当PEI与DNA混合时质量比在2:1及以上、转染时的DNA浓度等于或高于2 μg/mL、转染6 h及以上时可显著提高质粒转染效率；而混合时的DNA浓度和混合时间对转染效率的影响较小，其主要影响外源基因在细胞中的表达。在HEK293T细胞培养中，在5.0×104 cells/cm2的高接种密度下细胞在固定床载体表面均匀分布并较快进入指数生长期；而转染时过高或过低的细胞密度均不利于慢病毒生产，当细胞密度为1.0×106 cells/cm2时，可获得较高的慢病毒滴度。采用上述优化的工艺条件，在表面积为2.0 m2的国产固定床反应器中收获的慢病毒产量达2.4×1010 TU。本研究结果为建立基于固定床反应器的慢病毒载体高效生产工艺提供数据支撑。

关键词: 固定床生物反应器, 慢病毒, 转染条件, 细胞培养参数