Host Cell Proteins
DNA Helix Immortal

Human Embryonic Kidney (HEK293) Cells

HEK cells are the abbreviated term for “Human Embryonic Kidney” cells. This cell line is also referred to as HEK-293 or 293 cells. Frank Graham, who established this cell line, used to number his experiments; the original HEK 293 cell clone was from his 293rd experiment, hence the full name HEK293. HEK 293 cells were generated by transfection of cultures of normal human embryonic kidney cells with sheared adenovirus 5 DNA.

HEK 293 cells have been used for many years in cell biological research as well as expression system for therapeutic proteins and viruses for gene therapy. HEK 293 immortal cell line that is comparatively easy to handle. An immortalised cell line is a population of cells from a multicellular organism which would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division.1,2

Methods for generating immortalised cell lines

  • Isolation from a naturally occurring cancer. This is the original method for generating an immortalised cell line for example human HeLa cells.
  • Introduction of a viral gene that partially deregulates the cell cycle (e.g., the adenovirus type 5 E1 gene was used to immortalise the HEK 293 cell line; the Epstein-Barr virus can immortalise B lymphocytes by infection).
  • Artificial expression of key proteins required for immortality, for example telomerase which prevents degradation of chromosome ends during DNA replication in eukaryotes
  • Hybridoma technology, specifically used for the generation of immortalised antibody-producing B cell lines, where an antibody-producing B cell is fused with a myeloma (B cell cancer) cell.

Advantages of the HEK293 Cell Line

As mentioned above, HEK 293 cells are  easy to handle straightforward to grow in culture and to transfect. HEK293 is the predominant cell line used for the  high throughput transient expression of recombinant proteins and a well-established system for the production of viral vectors.3

Similar to CHO cells, the mammalian HEK293 cell line provides a natural environment for expression of eukaryotic proteins. which enables correct human post translational modifications. Membrane proteins with various PTMs can be harvested via recombinant protein expression.

However, like in all protein expression systems, protein manufacturing and purification process of these products leaves the potential for impurities by host cell proteins (HCP). HCP impurities can result in adverse toxic or immunological reactions and therefore regulation authorities demand to reduce these impurities to the lowest levels practical.

High transfection rates allow transient HEK293 cell lines to be used for protein production. the advantage of high transfection rates through stable cell lines does not need to be exploited – transient transfections for protein production are not only quicker than the generation of stably transfected cell lines but the transient transfection process is also less labor intensive and highly automatable by liquid handling robotics 4.

Optimization of the Expression System HEK293

Efficient optimization of promoter and signal peptides further enhance proteins expression. The expression can be further optimized by changing transfection protocol and cultivation time5,6.

Another approach for optimization lays in the metablism of HEK293. Understanding metabolic requirements in HEK293 significantly increases productivity through optimization of cellular metabolism Exhausted or critical substrates can be replenished and eventually toxic metabolites removed.7

This enables high throughput screening of candidate proteins to accelerate the identification of lead molecules for use in clinical trials. Thus, HEK293 cells are suitable for early stage pipeline development as well as upscaling into recombinant antibody production on industry level. HEK293 allows for greater process consistency and control of the final product quality. It can be well cultivated in serum-free media which simplifies the harvesting process.

HEK293 Modification: The 293T Cell Line

HEK cells are particularly interesting for virology to be more precise in the propagation of adenoviral vectors. Key genes lacking in adenoviral vectors (typically, E1 and E3) can be expressed by HEK 293 cells. The widespread use of the 293T line is due to its transfectability by the various techniques, including calcium phosphate method, achieving efficiencies approaching 100%. 293T cells, additionally expresses the SV40 large T antigen, which enables DNA replication of episomal plasmids with the SV40 origin of replication. This allows for amplification of transfected plasmids and extended temporal expression of desired gene products. Thus, certain retroviruses, such as lentiviruses, or DNA viruses can be replicated in 293T cells.8

HEK293 Cell Line – Role as a Model Organism

Organisms used for protein expression are extensively researched, fully sequenced and are usually characterized by their ease of use. Like  P. pastoris or E.Coli are often suited to serve as model organism.

HEK 293 cells cannot fullfill this role. Decades of 293 and 293-derivative cell cultivation have led to progressively different genome structure and sequence alterations. Knowledge of the genome sequence of 293 cells is not only important to understand basic 293 biology and the genomic changes associated with cell culture and clone selection, but is also especially relevant in the light of mammalian cell genetic engineering.

Furthermore it is not known whether a fibroblastic, endothelial, epithelial or some other type of cell have been used prior to transformation with adenovirus 5, because the cells were not properly characterized. TThis is why, HEK293 cannot be used as an in vitro model for kidney cell studies.

 

References

  1. Tom R, Bisson L, Durocher Y. (2008) . “Culture of HEK293-EBNA1 Cells for Production of Recombinant Proteins.” in  “CSH Protoc“.  prot 4976, PMID
  2. Arena TA, Harms PD, Wong AW. (2018) “High Throughput Transfection of HEK293 Cells for Transient Protein Production.”. in Methods Mol Biol 1850:179-187.
  3. Pieprzyk J, Pazicky S, Löw C. (2018) “Transient Expression of Recombinant Membrane-eGFP Fusion Proteins in HEK293 Cells.” in Methods Mol Biol 1850: 17-31. PMID
  4. Baldi L1, Muller N, Picasso S, Jacquet R, Girard P, Thanh HP, Derow E, Wurm FM. (2005) “Transient gene expression in suspension HEK-293 cells: application to large-scale protein production.”. in Biotechnol Prog 21(1):148-53. PMID
  5. Román R, Miret J, Scalia F, Casablancas A, Lecina M, Cairó JJ. (2016). “Enhancing heterologous protein expression and secretion in HEK293 cells by means of combination of CMV promoter and IFNα2 signal peptide.”  in J Biotechnol 10;239:57-60. PMID
  6. Thomas, Philip; et al. ,  (2005). “Optimization and automation of an end-to-end high throughput microscale transient protein production process.” in Biotechnol Bioeng 112:1832–1842.  DOI
  7.  Petiot E. (2015). “Influence of HEK293 metabolism on the production of viral vectors and vaccine.” in Vaccine. 4;33(44):5974-81
  8. Mao Y et al (2015). “Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells.” in Int J Med Sci. 15;12(5):407-15. PMID
  9. Daly R, Hearn MT (2005). “HEK293 cell line: A vehicle for the expression of recombinant proteins”. Journal of Pharmacological and Toxicological Methods. 51 (3): 187–200. PMID