293T细胞生产慢病毒工艺优化  

Optimization of Lentivirus Production Process for 293T Cells

作　　者：李欣 高驰 顾力行 曾毅 姚頔 何红鹏[1] 张同存[1,2] LI Xin;GAO Chi;GU Li-Xing;ZENG Yi;YAO Di;HE Hong-Peng;ZHANG Tong-Cun(Tianjin University of Science and Technology,School of Bioengineering,Department of Pharmacy,Tianjin 300457,China;Wuhan University of Science and Technology,College of Life Science and Health,Department of Biology,Wuhan 430081,Hubei,China)

机构地区：[1]天津科技大学生物工程学院药学系,天津300457 [2]武汉科技大学生命科学与健康学院生物学系,武汉430081

出　　处：《中国生物化学与分子生物学报》2024年第4期554-564,共11页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家重点研发计划(No.2018YFA0901700)资助。

摘　　要：随着细胞治疗的快速发展,大规模慢病毒的生产成为工艺环节中的瓶颈,因此,优化293T高滴度和高纯度的CAR慢病毒载体生产工艺显得至关重要。本研究旨在优化包装慢病毒的293T贴壁细胞,节省时间,节约成本,提高慢病毒包装的能力。同时对优化慢病毒载体中出现悬浮细胞结团生长的现象进行探索,检测其影响结团的因素。分别采用快速、慢速驯化方式将293T贴壁细胞驯化为悬浮培养,并比较其细胞形态、细胞密度、细胞活率、慢病毒包装能力和冻存复苏后稳定一致性,筛选出最优的悬浮驯化条件。通过调节Ca^(2+)浓度和EDTA添加量来研究比较细胞结团生长状况。结果证明,使用无血清培养基OPM-293 CD05溶剂(medium)可以将293T贴壁细胞快速驯化为293T悬浮细胞,并能制备出慢病毒滴度且优于贴壁细胞的包装滴度(^(*)P<0.05)。Ca^(2+)浓度会影响细胞结团大小,添加EDTA能有效分离分散非必要的细胞抱团生长。研究结果显示,传统293T贴壁细胞可以使用无血清培养基OPM-293 CD05溶剂快速驯化成悬浮细胞;在一定范围内,Ca^(2+)浓度越高细胞所结团块及粒径越大,EDTA添加量越高细胞所结团块及粒径变小。这为优化慢病毒载体包装工艺和悬浮培养条件,同时为体外规模化细胞培养放大和生产奠定了理论基础,具有一定的实用价值。With the rapid development of cell therapy,large-scale lentiviral production has become a bottleneck in the process chain;thus,optimizing the production process of CAR lentiviral vectors with high titer and high purity of 293T becomes crucial.This study aimed to optimize the packaging of lentiviral 293T adherent cells to achieve time-saving,cost-saving,and improved lentiviral packaging.At the same time,the optimization of lentiviral vectors was carried out to explore the factors affecting the growth of suspension cell clusters.The 293T adherent cells were domesticated into suspension culture by fast and slow domestication.Their cell morphology,cell density,cell viability,lentiviral packaging ability,and stable consistency after cryopreservation and resuscitation were compared to screen out the optimal suspension domestication conditions.Comparative cell clumping growth was studied by adjusting Ca^(2+)concentration and EDTA addition.The findings showed that the serum-free medium OPM-293 CD05 Medium could be quickly domesticated into 293T suspension cells from 293T adherent cells,and that these suspension cells could be produced with lentiviral titers that were better than the adherent cells’packaging titers(^(*)P<0.05).Ca^(2+)concentration affects the size of cell clusters.The addition of EDTA effectively separates and disperses unnecessary cell clusters.In summary,the experiment’s findings demonstrated that serum-free OPM-293 CD05 Medium could quickly domesticate conventional 293T adherent cells into suspension cells.Within a certain range,the higher the concentration of Ca^(2+),the larger the agglomerates and particle size,and the higher the addition of EDTA,the smaller the agglomerates and particle size.This provides a theoretical framework for the optimization of suspension culture conditions and the lentiviral vector packaging process.It also establishes a theoretical framework for the scale-up and manufacturing of in vitro cell culture,which has some practical applications.

关 键 词：慢病毒载体 293T细胞 悬浮驯化 悬浮细胞培养 结团 

分 类 号：Q813.1[生物学—生物工程]