Iron (III) citrate inhibits polyethylenimine-mediated transient transfection of Chinese hamster ovary cells in serum-free medium
Iron (III) citrate inhibits polyethylenimine-mediated transient transfection of Chinese hamster ovary cells in serum-free medium
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DOI：
10.1007/s10616-009-9198-8
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发表时间：
2009-07-01
期刊：
影响因子：
2.2
通讯作者：
Liu, Zhong
中科院分区：
文献类型：
--
作者：
Eberhardy, Scott R.;Radzniak, Laurie;Liu, Zhong
使用聚乙烯亚胺（PEI）作为转染试剂的瞬时转染方案的最新进展已经导致了经济方法的发展，所述经济方法提供了足以用于许多临床前需要的蛋白质的工业生产的产率。有许多变量可以优化以提高瞬时转染中的蛋白质表达，其中最关键的变量之一是细胞生长的培养基。虽然用PEI转染在含有血清的培养基中效果良好，但生物制药工业正在远离培养基中的动物源性组分。已经发现许多无血清培养基允许瞬时转染，但许多其他培养基不允许瞬时转染，原因尚不清楚。因此，了解无血清培养基中可抑制PEI介导的瞬时转染的组分将有助于培养基的开发。在本研究中，对用于中国仓鼠卵巢细胞的无血清培养基的各种组分进行了分析，其中PEI介导的瞬时转染被抑制。我们发现，添加到培养基中的铁补充剂是负责的抑制。进一步的研究表明，柠檬酸铁（III），一种常见的铁螯合剂，在无血清培养基中发现，是引起这种效果的特定成分。此外，我们表明，瞬时转染的抑制是由铁（III）柠檬酸盐，而不是柠檬酸盐或铁单独。最后，我们表明，在无血清培养基中的铁螯合剂，而不是铁（III）柠檬酸盐不抑制抗体的表达。
Recent advances in transient transfection protocols using polyethylenimine (PEI) as a transfection reagent have led to the development of economical methods that provide yields sufficient for industrial production of proteins for many preclinical needs. There are many variables that can be optimized to improve protein expression in transient transfection, and one of the most critical is the medium in which the cells are grown. While transfection with PEI works well in media containing serum, the biopharmaceutical industry is moving away from animal-derived components in media. A number of serum-free media have been found to allow transient transfection, but many others do not for reasons that are not clear. Thus, knowledge of the components of serum-free media that can cause inhibition of PEI-mediated transient transfection would be useful for media development. In this study, an analysis was performed of various components of a serum-free medium used for Chinese hamster ovary cells in which PEI-mediated transient transfection was inhibited. We found that an iron supplement added to the medium was responsible for the inhibition. Further investigation showed that iron (III) citrate, a common iron chelator found in serum-free medium, was the specific component that caused the effect. Further, we showed that inhibition of transient transfection was caused by iron (III) citrate specifically, rather than citrate or iron alone. Finally, we showed that various iron chelators in serum-free media other than iron (III) citrate do not inhibit antibody expression.