Gene therapy is a rapidly developing field. The most widely used technique for foreign gene transfer is lentiviral-mediated gene therapy. Lentiviral vector has been developed from the first generation to the third generation in terms of safety. The preparation of lentiviruses with high titer remains difficult. In this study, a Fibra-Cel sheet carrier was used as an HEK293T cell carrier matrix, and several sterile cell culture spinners were combined and cultured on a roller bottle machine to scale up the adherent cells. The virus titer was maximized by screening the factors to optimize the lentivirus titer in the third-generation lentivirus packaging process one by one. Fibra-Cel sheet vector was successfully used as the matrix of HEK293T cell adhesion to culture adherent cells at large scale. The optimal conditions for large-scale preparation of the third-generation lentivirus by bottle roller were screened and three batches of lentiviruses were produced on pilot scale. The production time of lentivirus was shortened from 120 hours to 54 hours from plasmid transfection to virus collection; in terms of cost, a rolling bottle machine was used instead of a bioreactor, leading to lower cost and no need for repeated sterilization during the whole process. The safe, effective and low-cost operation of successful production will provide a technical base for the large-scale preparation of lentivirus and thus lay a firm foundation for its clinical application.
基因治疗是一个快速发展的领域，其中关于慢病毒介导的外源基因导入研究得最为广泛。随着研发技术的不断发展，在安全性方面慢病毒已经从第一代发展至第三代，而如何工程化获得高滴度慢病毒仍是当今技术一大瓶颈。运用Fibra-Cel 片状载体作为HEK293T 细胞载体基质，联合多个无菌细胞转瓶在滚瓶机上培养，对贴壁细胞进行规模放大化生产。通过对第三代慢病毒包装过程中影响慢病毒滴度的因素进行逐一筛选，使病毒滴度达到最优。研究结果成功运用Fibra-Cel 片状载体作为HEK293T 细胞粘附载体基质，作为一种贴壁细胞规模放大的方法，筛选出了利用滚瓶机制备第三代慢病毒的最优条件，规模化生产了3 批慢病毒。在时间上，将慢病毒的生产时间从质粒转染到病毒收集的120 h 缩短至54 h；在成本上，利用滚瓶机代替生物反应器实现了无需反复灭菌、全程一次性产品的安全有效低成本的运行，为慢病毒的规模化制备提供了技术上的支持，为临床应用奠定了坚实的基础。.
Keywords: CAR-T; Fibra-Cel; gene therapy; large-scale preparation; lentivirus.