High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI
High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI
复制标题
DOI：
10.1002/bit.21596
复制
发表时间：
2008-02-15
影响因子：
3.8
通讯作者：
Wurm, Florian M.
中科院分区：
文献类型：
--
作者：
Backliwal, Gaurav;Hildinger, Markus;Wurm, Florian M.
重组蛋白具有巨大的商业和科学意义。然而，在哺乳动物细胞中的大多数生产方法涉及创建稳定细胞系的耗时和费力的步骤。基于瞬时基因表达的生产方法在速度和通用性方面是有利的;然而，取决于转染方案，瞬时转染面临一些瓶颈，例如先验复合物形成、所用转染和生产介质方面的限制以及在转染之前和/或之后需要更换介质。公开的用聚乙烯亚胺转染悬浮适应的HEK-293细胞的方案在克服这些瓶颈中的一些方面显示出很大的希望，但仍然需要先验复合物形成以获得最佳产率，并限制了转染和生产培养基的选择。在这里，我们报告了成功的原位转染悬浮适应HEK-293细胞与25 kDa线性聚乙烯亚胺密度高达20 × 10（6）细胞/毫升，在复杂的培养基中，随后生产在较低的细胞密度（1 × 10（6）细胞/毫升）。在将细胞浓缩至如此高的密度后，HEK-293细胞的转染在最常用的培养基中变得可能，并且不限于特定的培养基。此外，不需要事先制备转染复合物，这是一个防止用于转染的DNA原液的在线无菌过滤的步骤，这是将工艺规模扩大到100或1，000 L时的一个重要考虑因素。最后，在复合培养基中以高密度转染HEK-293细胞上级现有的转染方案，并且使通过瞬时基因表达可获得的重组蛋白的产量加倍。
Recombinant proteins are of great commercial and scientific interest. Yet, most production methods in mammalian cells involve the time- and labor-consuming step of creating stable cell lines. Production methods based on transient gene expression are advantageous in terms of speed and versatility; yet, depending on the transfection protocol, transient transfection faces some bottlenecks such as a priori complex formation, limitations in terms of transfection and production media used and the need for medium exchange prior to and/or after transfection. Published protocols for transfection of suspension-adapted HEK-293 cells with polyethyleneimine have shown great promise in overcoming some of these bottlenecks, but still require a priori complex formation for optimal yields and limit the choice of transfection and production media. Here, we report successful in situ transfection of suspension-adapted HEK-293 cells with 25-kDa linear polyethyleneimine at densities up to 20 x 10(6) cells/ml, in complex media followed by production at lower cell densities (1 x 10(6) cells/mL). After concentrating cells to such high densities, transfection of HEK-293 cells becomes possible in most commonly used media and is not restricted to a specific medium. Furthermore, there is no need to make transfection complexes a priori, a step that prevents inline sterile filtration of the DNA bulk for transfection, an important consideration when scaling processes up to 100 or 1,000 L. Finally, transfecting HEK-293 cells at high density in complex media is superior to existing transfection protocols and doubles yields of recombinant protein obtainable by transient gene expression.