High Throughput Transfection of HEK293 Cells for Transient Protein Production

Methods Mol Biol. 2018;1850:179-187. doi: 10.1007/978-1-4939-8730-6_12.


Transient transfection of mammalian cells is used in the biotechnology industry to quickly supply recombinant protein for research and large molecule drug development. Here, we describe a method for high throughput transient transfection of Human Embryonic Kidney 293 (HEK293) cells in 30 mL tubespins using polyethylenimine (PEI) as a transfection reagent. An automated liquid handler can be used to perform pipetting steps for transfecting batches of 96 tubespins, and septa in the tubespin caps allow for rapid processing without decapping. The addition of valproic acid (VPA) to transfection cultures enhances recombinant protein production. The thawing and passaging operations for HEK293 cultures to source the transient transfections are also described.

Keywords: Automation; HEK293 cells; High throughput; Polyethylenimine; Recombinant protein production; Transient transfection.

MeSH terms

  • Biotechnology
  • HEK293 Cells
  • High-Throughput Screening Assays / methods
  • Humans
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*
  • Transfection / methods*


  • Recombinant Proteins