- Large-scale transient transfection of mammalian cells in suspension is a widely used method for the rapid production of recombinant proteins, particularly in preclinical research, assay development, and early-stage biomanufacturing.
- The technique allows for the short-term expression of a gene of interest without genomic integration, enabling the generation of milligram to gram quantities of protein within a few days.
- Suspension cultures of mammalian cells—most commonly HEK293 and CHO cells—are favored for their ability to grow in serum-free, chemically defined media and to perform complex post-translational modifications such as glycosylation, disulfide bonding, and protein folding that are critical for therapeutic relevance.
- The history of transient transfection dates back to the early 1980s, when calcium phosphate-mediated DNA uptake into adherent cells was first reported. However, the method was inefficient and unsuitable for scaling.
- A major breakthrough came in the late 1990s to early 2000s, with the development of polyethylenimine (PEI)-based transfection protocols, which enabled high-efficiency DNA delivery in suspension cultures. Around the same time, HEK293-derived cell lines such as 293F and 293T, adapted for serum-free suspension growth, became widely used in research and industrial settings. The ability to scale transient transfection from milliliter cultures in shake flasks to liter-scale and beyond in wave bags and stirred-tank bioreactors opened the door for rapid, cost-effective production of functional recombinant proteins, antibodies, and virus-like particles.
- Modern transient transfection platforms benefit from optimized vectors, high-density cell culture systems, and improved DNA-to-reagent formulations. These innovations have made transient expression a reliable alternative to stable cell line development for short-term protein production needs, particularly in settings where speed and flexibility are paramount. Today, large-scale transient transfection plays a vital role in early drug discovery, vaccine development, and the production of materials for structural biology and functional studies.
