HEK293 Cells

HEK293 Cells: The Essential Guide for Researchers

Human embryonic kidney 293 (HEK293) cells are a human embryonic kidney cell line that has gained widespread popularity in the scientific community due to their versatility and usefulness in a wide range of research applications. The cell line was established in the early 1970s and has since been used for vaccine development, cancer research, drug testing, and signal transduction. This blog post will explore all aspects of the HEK293 cell line, including its origin, culture information, advantages and drawbacks, applications, and resources.

HEK293 Cells: General Information and Origin

What are HEK293 cells?

HEK293 cells are a human embryonic kidney cell line derived from the kidney tissue of an electively terminated human embryo of unknown parenthood. The cells were established by a Dutch biologist named Alex Van der Eb in the early 1970s. They were later immortalized through a transformation with a truncated adenovirus 5 by researcher Frank Graham.

Initially, the cells seemed challenging to transform. However, after many continuous efforts, cell growth happened from an isolated single transformed clone [1]. The cell's transfection with adenovirus 5 led to the inclusion of the E1A and E1B genes in the cell's genome, which prevents cell death and allows for abundant protein production. Before immortalization, the fetal kidney cells were not adequately characterized, so their exact cell type is unknown.

Embryonic kidneys are composed of endothelial, epithelial, and fibroblast cells, so HEK293 cells likely belong to these cells. However, the mRNA and gene products suggest that they are neuronal cells. It is possible that the addition of Ad5 altered the cellular phenotype and gene expression. Fun fact: The "293" in HEK293 refers to the 293rd experiment performed by Graham.

Fun fact: The "293" in HEK293 refers to the 293rd experiment performed by Graham.

Characteristics of HEK293 cells

  • Morphology
  • Cell size
  • Genome and ploidy (chromosome number)

HEK293 cells have a shape that resembles epithelial cells. Embryonic kidneys are composed mainly of fibroblast, endothelial, and epithelial cells. Thus 293 cells resemble one of these cell types in shape.

HEK293 cells in culture

The size of HEK293 cells falls between 11 to 15 µm, which can be affected by the culture conditions. In culture, the cells may appear flattened when grown on a surface or rounded in suspension. HEK293 cells are hypotriploid, and about 30% of HEK293 cells have a modal ploidy of 64 chromosomes, but some cells have even more chromosomes. The cells also have three copies of the X chromosome and a 4-kilobase pair fragment of adenovirus 5 integrated into chromosome 19.

Comparison of HEK293 Vs. HEK293T cell line

Many derivatives have been derived from HEK293 cells, including HEK293T and HEK293F cells. HEK293T cells are one of the most widely used derivatives and were created by incorporating a temperature-sensitive SV40 T-antigen mutant into the original HEK293 cell genome. Expression of the T-antigen allows for the replication of plasmids with an SV40 origin of replication when transfected into 293-T cells, leading to increased production of recombinant proteins [2]. For more information on HEK293 cell line derivatives, including their development and characteristics, refer to this review article.

Basics of HEK293 Cell Culturing: A Step-by-Step Guide



Population Doubling Time

The doubling time of the HEK293 cell line ranges from 24 to 45 hours, with an average of 30 hours.

Adherent or Suspension Cultures

HEK293 cells can be grown in both adherent and suspension forms. Adherent cells grow as monolayers, while suspension cultures grow as spheroids.

Seeding Density

Split cells at 80-90% confluency during the growth phase. Detach cells using Accutase and seed at a density of 1 to 4 x 104 cells/cm2. A confluent layer will form in 4 days at a seeding density of 1 x 104 cells/cm2.

Growth Medium

Grow in Eagle's Minimum Essential Medium (EMEM) with 2 mM L-glutamine and 10% fetal bovine serum (FBS). Change the medium two times a week.

Growth Conditions (Temperature, CO2)

Keep in a humidified incubator at 37 °C with a 5% CO2 supply for optimum growth.


Store in the vapor or liquid phase of liquid nitrogen for long-term preservation. Avoid storing at -80 °C freezer as it may affect cell viability.

Freezing Process and Medium

Use a slow freezing method for best preservation. Freeze in CM-1 or CM-ACF freeze medium available from CLS.

Thawing Process

Thaw frozen cells in a 37 °C water bath for 1-2 minutes until a small ice clump remains. Transfer the cell suspension to a centrifuge tube and add pre-warmed growth medium and centrifuge to eliminate freezing media components. Resuspend the cell pellet in fresh medium and culture at optimal conditions.

Biosafety Level

HEK293 cells require biosafety level 1 handling.


The HEK293 Cell Line in Research & Industry

The human embryonic kidney cell line has a wide range of applications in research. Here we will discuss a few research areas utilizing 293 cells. The pharmaceutical industry uses HEK293 cells for high-throughput screening assays because they can be transfected with plasmids encoding drug targets and grown in large numbers. In industrial biotechnology, HEK293 cells are used to produce recombinant proteins and enzymes and to develop biosensors. Toxicology researchers, in turn, use HEK293 cells to study the effects of chemicals and environmental toxins on cellular processes and gene expression. HEK293 cells are also used to produce recombinant viruses such as adenoviruses and lentiviruses for gene therapy, viral replication and host-virus interaction.

  1. Protein production: HEK293 cells are suitable for large-scale protein and therapeutic vaccine manufacture. The cell line is further used to generate viral vectors such as adeno associated and adenoviral vectors [3, 4]. Recently, HEK293 cells have been used for producing a crucial recombinant protein, erythropoietin (EPO) [5].
  2. Receptor signaling: HEK293 cells are used for heterologous expression of various cell membrane ion channels and receptors. They have been used to study G protein-coupled receptor signaling pathways in diseases.
  3. Drug testing: HEK293 cells are frequently used for testing the toxicity of drugs and natural products [6, 7].
  4. Cancer research: 293 cells are tumorigenic, and crucial gene expression changes can aggravate tumorigenesis in this cell line. Therefore, the 293 cell line is frequently used in cancer studies for understanding the underlying molecular mechanisms and drug development [8].

Table 2: Selected research publications on the use of HEK293 cells

Publication Title


Study goal

Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin

Journal of Biotechnology

The study used HEK293 cells to develop a high cell-density perfusion method for recombinant human Erythropoietin protein production.

Zika virus-like particles (VLPs): Stable cell lines and continuous perfusion processes as a new potential vaccine manufacturing platform


The publication reported using human embryonic kidney cells to develop a stable cell line expressing Zika viral-like particles (VLPs), which can be promising Zika vaccine candidates and are produced mainly by growing stable cells in stirred-tank perfusion bioreactors.

Plasticity of the HEK- 293 cells, related to the culture media, as platform to produce a subunit vaccine against classical swine fever virus

AMB express

Researchers developed a HEK293 cell line expressing classical swine fever virus E2 glycoprotein fused to the porcine CD154 and compared these cells' growth and expression profiles in four commercially accessible growth media.

Cytotoxicity of apigenin toward multiple myeloma cell lines and suppression of iNOS and COX-2 expression in STAT1-transfected HEK293 cells


HEK293 cells were transfected with the STAT-1 gene to develop a model for investigating the role of the STAT1/COX-2/iNOS signaling pathway in tumorigenesis and inflammation, and the inhibitory potential of apigenin was explored against this pathway.

Feasible development of stable HEK293 clones by CRISPR/Cas9-mediated site-specific integration for biopharmaceuticals production

Biotechnology Letters

The authors reported CRISPR/Cas9 technique as an efficient and reliable tool for developing stable cell lines by site-specific integration.

Dissecting the roles of GRK2 and GRK3 in μ-opioid receptor internalization and β-arrestin2 recruitment using CRISPR/Cas9-edited HEK293 cells

Scientific Reports

The publication used CRISPR-Cas9 technology for genome editing of HEK293 cells and knocked out G-protein coupled receptor-kinases GRK2 and GRK3 to study their impact on µ-opioid receptor internalization and recruitment of β-arrestin.


HEK293 Subculture Protocol

Required Reagents
  1. 1X Phosphate-Buffered Saline (PBS)
  2. 10% Trypsin-PBS
  3. Dulbecco's Modified Eagle's Medium (DMEM)

Cell Preparation

  1. Check the HEK cells under a microscope to ensure they are about 90% confluent.
  2. Clean the workstation using aseptic techniques and sterilize the fume hood with UV light.
  3. Wipe the workspace with 70% ethanol.
  4. Pre-warm all reagents in a 37°C water bath.

Calculating Split Fraction and Seed Amount

  1. Determine the split fraction, typically between 1:5 to 1:20.
  2. Calculate the volume for pipetting using the formula: Vp = (S)(Vd).


Growth Area (cm^2)

Media Needed (mL)

Ratio to 100 mm Dish

6 well plate




100 mm plate




100 cm2 flask




150 mm plate




Splitting the Cells

  1. Remove the old media using an aspirating pipette.
  2. Wash the cells with 10 mL 1X PBS.
  3. Incubate the cells with Accutase for around 10 minutes.
  4. Neutralize the trypsin with 1 mL of DMEM.
  5. Centrifuge the cells for 2 to 3 minutes at 300 g.
  6. Resuspend the cells in 10 mL of DMEM.
  7. Count the cells or calculate the appropriate dilution based on confluence.
  8. Seed the cells onto a new plate and incubate.

Confluent HEK293 cells cultured at CLS 

Benefits and Limitations of the HEK293 Cell Line

HEK293 cells have distinctive features that make them attractive for research and protein production.


  • High Recombinant Protein Production: HEK293 cells can produce large amounts of recombinant proteins with complex post-translational modifications.
  • Flexible Transfection: These cells are highly efficient for transfection studies and can be efficiently transfected using various physical and chemical methods.
  • Gene Expression Analysis: Due to their ability to be efficiently transfected, HEK293 cells can be used for both transient and stable gene expression analysis.
  • Reproducibility of Results: HEK293 cells offer consistent, reliable, reproducible results, making them a popular choice for research laboratories.


Drawbacks of the HEK293 Cell Line

  • Bacterial contamination: The risk of bacterial contamination is a common challenge in culturing cell lines, including HEK293 cells. Bacterial infections can alter the pH of the culture media, cause turbidity, and affect cell shape, culture period, and gene expression. To prevent contamination, aseptic cell culturing conditions must be strictly maintained.
  • Viral infection: HEK293 cells, like other human cell lines, are susceptible to human viral diseases. These infections can only be detected through PCR testing and are not easily visible.
  • Culture period: Although the HEK293 cell line is immortalized, prolonged culture periods can gradually degrade cell health and affect gene expression, reproducibility, and cell growth. To maintain a healthy culture, it's recommended to keep the number of passages below 20.

HEK293 Resource Overview: Protocols, Videos, and More

HEK293 cells are a widely used and well-studied cell line, leading to various resources for their maintenance and cultivation. Here, we highlight some resources for learning about HEK293 cell culture protocols:

Videos related to HEK293 cell line

Many educational videos are readily available on subculturing, cell plating, and transfection protocols of HEK293 cells.

Unleash the potential of your research with HEK293 cells! We've got you covered with all the information you need to get started, so why wait? Make the smart choice and order from us today to experience the benefits of using this incredible cell line in your study!

Reference list

  1. Lin, Y.-C., et al., Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations. Nature communications, 2014. 5(1): p. 4767.
  2. Tan, E., et al., HEK293 cell line as a platform to produce recombinant proteins and viral vectors. Frontiers in bioengineering and biotechnology, 2021: p. 1288.
  3. Pulix, M., et al., Molecular characterization of HEK293 cells as emerging versatile cell factories. Current Opinion in Biotechnology, 2021. 71: p. 18-24.
  4. Alvim, R.G., I. Itabaiana Jr, and L.R. Castilho, Zika virus-like particles (VLPs): Stable cell lines and continuous perfusion processes as a new potential vaccine manufacturing platform. Vaccine, 2019. 37(47): p. 6970-6977.
  5. Schwarz, H., et al., Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin. Journal of biotechnology, 2020. 309: p. 44-52.
  6. Liu, X., et al., Nanotoxic effects of silver nanoparticles on normal HEK-293 cells in comparison to cancerous HeLa cell line. International journal of nanomedicine, 2021. 16: p. 753.
  7. Patra, B., et al., Piper betle: augmented synthesis of gold nanoparticles and its in-vitro cytotoxicity assessment on HeLa and HEK293 cells. Journal of Cluster Science, 2020. 31: p. 133-145.
  8. Stepanenko, A. and V. Dmitrenko, HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution. Gene, 2015. 569(2): p. 182-190.