HEK cells: Origin, structure and use of human embryonic kidney cells in research

Nowadays, it is impossible to imagine research without HEK cells. Work with the human kidney cell line has already led to many new findings in various fields of research. They are used in virology and pharmacy, for example, but also play a major role in cell research. In research, HEK cells are now just as important as HeLa cells. One type of HEK cell in particular has become established.

 Learn more in the following text about the topics mentioned:

  • What are HEK cells and how are they constructed?
  • How are they used in research?
  • What are the challenges in analysis and how can fluidlab support?

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What are HEK cells

HEK cells are human embryonic kidney cells, which were isolated in the 1970s. Transformation of HEK cells with DNA parts of human adenovirus 5 resulted in the cell line HEK293. The original HEK cell line is contaminated with HeLa cells, making it unusable.1 2 The cells are known to be easy to culture and transfect, which is why they are often used in cancer research.

In addition, their high transfection efficiency, the production of exogenous proteins or viruses, makes them highly useful for pharmaceutical and biomedical research purposes.

The cells are used for the development of viral vaccines and chemotherapeutics or to produce different types of vectors: Adenoviruses, Lentiviruses or AAV vectors.2 3

The structure of a HEK cell

HEK cells are constructed like other eukaryotic cells. They grow adherently and are hypotriploid cells, as there are less than three times the number of chromosomes as in human gametes. .

 During the development of HEK cells, 4.5 kilobases of the adenovirus 5 genome were incorporated into the cells by transformation.

 Accordingly, their karyotype is more complex, as they contain two or even more copies of each chromosome. Thus, there are three copies of the X chromosome and four copies each of chromosomes 17 and 22. Initially, HEK cells were thought to be epithelial cells of the kidney, however, due to their partly similar characteristics to nerve cells, it is more likely that these are precursor cells of the adrenal gland.4

The importance of human kidney cells for research

Due to the ease of cultivation and transfection, HEK cells are very popular in research. They are suitable for experiments that focus on the behavior of components within the cell. This may involve processes within the cell or cell compartments.5

HEK293 cells are used to produce adenoviral and adeno-associated viral (AAV) vectors, as previously mentioned, because they contain the adenoviral E1A/B genes, which play a helper role in the production of viral vectors.6 Adenovirus 5 is an important vector used in gene therapy. This virus is also a common cause of acute respiratory infections.5

The 293T (originally 293tsA1609neo) cells, which are a derivative of HEK293 cells, are particularly popular in virology because they also express the SV large T antigen, which makes them highly transfectable. The T antigen enables retroviruses or even DNA viruses to replicate in these cells, which is particularly important for basic research into viral mechanisms.7

For example, the cells play an important role in the treatment of rabies diseases. Rabies viruses can cause fatal encephalitis, a dangerous inflammation of the brain, which is why rapid diagnosis, and, for this purpose, rapid virus isolation is necessary. This allows effective measures to be subsequently initiated in infected individuals and epidemiological surveillance to be controlled.

 Madhusudana et al. 2010 demonstrated that HEK293 cells are particularly sensitive to neurotropic virus because they express multiple neuronal proteins, which would be beneficial for rapid isolation and diagnosis.7

In a recent study by Horibata et al. 2021, HEK cells were used to test whether Zika virus, which is also neurotropic, is capable of infecting and replicating these cells. HEK293 cells demonstrated that Zika virus infection leads to impaired antiviral and inflammatory responses, which alter host gene expression.8

Apart from virology, HEK cells also play a significant role in cell research. For example, HEK cells were used in Balatti et al. 2017 to create a knocked-out cell model with ts-101 and ts-46. These are clusters of small RNAs derived from transfer RNAs. It has been demonstrated using HEK cells that certain RNAs formed during the maturation process of tRNAs are dysregulated in cancer. 

Using the cell model with HEK cells, it was found that significant differences, between infected and uninfected cells, exist in gene expression patterns.

On the one hand, there was an activation of genes involved in cell survival. On the other hand, a silencing of genes involved in cell death and in the production of chromatin structure could be detected.9

HEK cells are also used in the development of drugs. In this context, biotherapeutic proteins, which play an important role in the treatment of autoimmune diseases, hormonal disorders, cancer, and other diseases, are of particular importance. These proteins can be synthesized with the help of HEK cells.

These proteins are now produced from human cell lines, such as HEK cells, because this is more likely to result in a recombinant protein that has post-translational modifications and thus matches endogenous human proteins. This is not always the case with other mammalian cell lines.

HEK293 cells are among the most widely used human cell lines to produce biotherapeutic proteins. Recently, five compounds produced in HEK293 cells have been approved for therapeutic use.

One of these is Drotecogin alfa, a recombinant activated protein C is used to treat patients with severe sepsis.10

The analysis of HEK cells in the lab

Working with HEK cells proves to be particularly easy in most cases and is done using standard procedures. Nevertheless, it is important to consider some aspects. HEK cells are among the most frequently analyzed cell lines.

HEK cells are easy to culture in a humidified incubator at 37°C and 5% CO₂. They can be cultivated not only as monolayers but also in suspension.

In addition, HEK cells are very easy to transfect using a variety of methods. A classical transfection method is the calcium-phosphate procedure. Here, a DNA-phosphate complex is precipitated onto the cell membrane. Other well-known methods are electroporation or the use of DEAE-dextran. Special kits for transfection of HEK cells further simplify the process.

Due to their rapid growth, they can be passaged every few days by a standard procedure. However, for this they should be in the logarithmic phase and thus have a confluence of <100%. Therefore, it is also important to pay attention to the number of cells that are seeded. For this purpose, automated cell counters can be used, or counting can be done manually by means of a counting chamber.

They are particularly attractive for the production of proteins because they are able to produce them in large quantities.

Further advantages of HEK cells are their high resistance and their semi-hard and low-maintenance growth. They can be used not only for stable but also for transient expression of proteins. Thus, they can perform post-translational folding and processes, which are necessary to produce mature proteins. 11


Challenges in the analysis of renal cells

Although HEK cells have many advantages, there are also some challenges that need to be considered when working with the cells. For example, it should be noted that culture of the cells for extended periods of time can lead to poorer cell health. This can have an impact on growth rate and translational efficiency, negatively affecting the reliability of experimental results where 'old cells' were used. Therefore, it is recommended to culture the cells only up to the 20th passage.

Another challenge is the risk of contamination in human cell lines. For this reason, sterile work with such cells is of particular importance.10

In general, when measuring viability of HEK cells, it should be noted that the cell line, compared with others, is less round and the morphology of the cells appears less uniform.

In addition, counting can be complicated by cell clusters, which may occur frequently due to cell adherence.4

The fluidlab offers these advantages for cell analysis

The neural network of the fluidlab analyzes and detects cells. Using the machine learning method, it not only differentiates living from dead cells, but is also able to distinguish cells and other particles in the sample. This has the advantage that even HEK cells, which do not constantly show a uniform morphology, can be detected by the device. It is also able to detect and count individual cells within a cell cluster, which can be of great advantage with HEK cells that tend to cluster.

The cells are analyzed without the use of a dye, which makes the viability measurement particularly gentle on the cells. With the aid of the fluidlab, it can be quickly and easily determined whether the viability rate of the HEK cells is high, and they can be used further or whether the passage is already too high.

The fluidlab R-300 has a very large field of view of 5.3 mm², by means of which a particularly high number of cells can be detected in the sample. This ensures a high accuracy of results, related to the viability and concentration of the initial sample.

In addition, the automated counting by the fluidlab, eliminates the human error and increases the precision. These two features make the statistical accuracy of the device very high.



1 Graham F. L., Russell W. C., Smiley J., Nairn R. (1977). Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5. J. Gen. Virol. 36, 59–72. 10.1099/0022-1317-36-1-59

2 Stacey, G. N., & Merten, O. W. (2011). Host cells and cell banking. Methods in molecular biology (Clifton, N.J.)737, 45–88.

3 Zur Hausen H. (1967). Induction of specific chromosomal aberrations by adenovirus type 12 in human embryonic kidney cells. Journal of virology1(6), 1174–1185.

4 Stepanenko, A. A., & Dmitrenko, V. V. (2015). HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution. Gene, 569(2), 182–190.

5 Tan, E., Chin, C., Lim, Z., & Ng, S. K. (2021). HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors. Frontiers in bioengineering and biotechnology9, 796991.

6 Montes-Galindo, D. A., Espiritu-Mojarro, A. C., Melnikov, V., Moy-López, N. A., Soriano-Hernandez, A. D., Galvan-Salazar, H. R., Guzman-Muñiz, J., Guzman-Esquivel, J., Martinez-Fierro, M. L., Rodriguez-Sanchez, I. P., Paz-Michel, B., Zaizar-Fregoso, S. A., Sanchez-Ramirez, C. A., Ramirez-Flores, M., & Delgado-Enciso, I. (2019). Adenovirus 5 produces obesity and adverse metabolic, morphological, and functional changes in the long term in animals fed a balanced diet or a high-fat diet: a study on hamsters. Archives of virology164(3), 775–786.

7 Madhusudana, S. N., Sundaramoorthy, S., & Ullas, P. T. (2010). Utility of human embryonic kidney cell line HEK-293 for rapid isolation of fixed and street rabies viruses: comparison with Neuro-2a and BHK-21 cell lines. International journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases, 14(12), e1067–e1071.

8 Horibata, S., Teramoto, T., Vijayarangan, N., Kuhn, S., Padmanabhan, R., Vasudevan, S., Gottesman, M., & Padmanabhan, R. (2021). Host gene expression modulated by Zika virus infection of human-293 cells. Virology, 552, 32–42.

9 Balatti, V., Nigita, G., Veneziano, D., Drusco, A., Stein, G. S., Messier, T. L., Farina, N. H., Lian, J. B., Tomasello, L., Liu, C. G., Palamarchuk, A., Hart, J. R., Bell, C., Carosi, M., Pescarmona, E., Perracchio, L., Diodoro, M., Russo, A., Antenucci, A., Visca Ciardi, P., Harris, C. C., Vogt, P.K., Pekarsky, Y,Croce, C. M. (2017). tsRNA signatures in cancer. Proceedings of the National Academy of Sciences of the United States of America, 114(30), 8071–8076

10 Dumont, J., Euwart, D., Mei, B., Estes, S., & Kshirsagar, R. (2016). Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Critical reviews in biotechnology, 36(6), 1110–1122.

11 Thomas P. and Smart T. (2005) HEK293 cell line: A vehicle for the expression of recombinant proteins. Journal of Pharmacological and Toxicological Methods, 51(3), 187-200.