High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI

Biotechnol Bioeng. 2008 Feb 15;99(3):721-7. doi: 10.1002/bit.21596.


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.

MeSH terms

  • Cell Line
  • DNA / chemistry*
  • DNA / genetics*
  • Drug Carriers / chemistry*
  • Humans
  • Kidney / physiology*
  • Polyethyleneimine / chemistry*
  • Protein Engineering / methods*
  • Recombinant Proteins / metabolism*
  • Transfection / methods*


  • Drug Carriers
  • Recombinant Proteins
  • Polyethyleneimine
  • DNA