HEK293T Cells: Unlocking the Potential in Transfection Studies

HEK293T cells are human embryonic kidney cells widely applied in industrial biotechnology, toxicology and cancer research. These immortalized cells are also used to produce a variety of therapeutic proteins and viruses.

In this article, we will mention all the imperative information about the HEK293 cell line, you need to start working with it, including:

General characteristics and origin of HEK293T cells
Culturing information of HEK293T cell line
HEK293T cell line: Advantages & Limitations
Research applications of HEK293T cell line
HEK293 cells: Research publications
Resources for HEK293T cell line: Protocols, Videos, and More

1. General characteristics and origin of HEK293T cells

This section of the article will cover the origin and general information of the HEK293T cell line. You will know: Where do HEK293T cells come from? What is the morphology of 293T cells? What is the HEK293 Cell Size?

HEK293T cell line originated from primary human embryonic kidney cells cultured in the laboratory. These cells were initially developed by transfecting embryonic kidney cells with sheared adenovirus type 5 DNA fragments in the early 1970s. HEK293T cell line was established by introducing simian virus 40 (SV40) large T-antigen to the HEK293 cells genome. This modification made 293 cells to be easily transfected and suitable for protein production and gene expression studies [1].
HEK293T cells possess an epithelial-like morphology. They represent an elongated and flattened shape with a definite cell boundary.
The size of HEK293T cells ranges between 11-15 µm in diameter.
The sv40 large t antigen HEK293T cells possess a complex karyotype. These cells are hypotriploid, containing 3 times less number of chromosomes than a haploid gamete. Moreover, they possess a modal chromosome number of 64.

What is the difference between HEK293T and HEK293?

Both HEK293 and HEK293T cell lines are of human origin. HEK293T is a popular derivative of the HEK293 cell line. These cells were developed from the original human embryonic kidney 293 cells by transfection with SV40 large T antigen. Whereas the immortalized HEK293 cells were established by the transformation and culturing of human embryonic kidney cells with sheared human adenovirus 5 DNA fragments.

Human embryonic kidney 293 cells observed under the microscope.

2. Culturing information of HEK293T cell line

HEK293T cells are extensively cultured in research laboratories. Before developing HEK293T cell culture, you must know: What is the doubling time of HEK293T cells? What is HEK293T media? What is HEK293T seeding density?

Key Points for Culturing HEK293T Cells

Doubling Time:	The doubling time reported for HEK293T cells is 30 hours.
Adherent or in Suspension:	HEK293T is an adherent cell line.
Seeding Density:	HEK293T cells are seeded at a density of 1 x 10⁴ cells/cm². At this seeding density, cells can make a confluent monolayer in approximately 4 days. For seeding, Adherent cells are detached by using the accutase dissociation solution. Detached cells are centrifuged and then carefully resuspended using a growth medium. Afterward, cells are dispensed into new flasks for culturing.
Growth Medium:	HEK293T cells are cultured in EMEM (Eagle's minimal essential medium) containing 1.0 g/L L-glucose, 2.2 g/L NaHCO3, 2.0 mM L-glutamine, and 10% fetal bovine serum. Media should be replaced 2 times a week.
Growth Conditions:	HEK293T cell cultures are kept in a 37°C humidified incubator with a 5% CO2 supply.
Storage:	Human embryonic kidney HEK293T cells are stored in the vapor phase of liquid nitrogen or at below -150°C temperature for longer terms.
Freezing Process and Medium:	HEK293 cells can be frozen in CM-1 or CM-ACF freezing media. Slow freezing processes that allow a gradual 1°C drop in temperature, are recommended for HEK293T cells to protect their viability.
Thawing Process:	Frozen cell vial is rapidly agitated in a water bath (37°C temperature) until a small ice clump is left. Cells are resuspended in media and centrifuged to eliminate components of freezing media. Recovered cells are then cultured in new flasks containing growth medium.
Biosafety Level:	Biosafety level 1 laboratory is required to handle and maintain HEK293T cell cultures.

Semiconfluent and confluent monolayers of HEK293T cells.

3. HEK293T cell line: Advantages & Limitations

A unique combination of advantages and limitations is associated with human embryonic kidney 293T cells. Here, we will go over a few main advantages and disadvantages of this cell line.

Advantages

The significant advantages of HEK293T cells are:

High transfectivity	HEK293T cell line exhibits high transfection efficiency. The cells efficiently uptake foreign DNA and produce abundant protein. They are commonly used for transient and stable transfection studies.
Easy to maintain	HEK293T cell cultures are easy to maintain and handle in research laboratories. They are considered an excellent choice for lab experiments.

Limitations

The limitation associated with HEK293T cell cultures are:

Contamination risk	HEK293T cells are susceptible to microbial contamination, which can impact cell morphology, gene expression, and other characteristics. This can lead to inaccurate experimental results.
Culture period	HEK293T cells can be used for long-term experiments. However, prolonged culture periods can compromise cell health and influence transfection efficiency and growth rate. Therefore, the passage number is limited to 20 or below.

4. Research applications of HEK293T cell line

Here, you will learn about some significant research applications of the HEK293T cell line.

Cancer research: HEK293T cell line is extensively used to study cancer progression, development, and underlying molecular mechanisms. Moreover, it is suitable for testing new cancer therapies. A study used the HEK293T cell line to investigate the role of the AMPKα1 gene in regulating lung and breast cancer progression. The researchers developed a knockout model (AMPKα1KO) and studied the gene function in detail [2].
Vaccine development: HEK293T cell line has been used to study viruses and produce viral vector-based vaccines to combat a variety of viral infections. A study used this embryonic kidney cell line for investigating the structural and functional Basis of COVID-19 virus cell entry through human Angiotensin-converting enzyme 2 (ACE2) [3]. In addition, a recent study used HEK93T cells to generate SARS-CoV-2 spike-pseudotyped lentivirus [4].
Toxicology research: This human embryonic kidney cell line is widely used to test the toxicity and efficacy of drugs. Such as research conducted in 2022 used HEK293T as a normal human cell line to validate the cytotoxic potential of Caladium lindenii extracts against liver cancer cell line, HepG2 [5].
Gene expression studies: The SV40 large T antigen HEK293T cell line is highly amenable to transfection, thus suitable for gene expression studies. A study used HEK293T cells to study the role of long noncoding RNA SNHG16 in regulating trophoblast functions. The study revealed that LncRNA SNHG16 interacts with the miR-218-5p/LASP1 axis to mediate these effects [6].

5. HEK293 cells: Research publications

In this section, we have mentioned a few promising research publications featuring HEK293T cells.

Development of SARS-CoV-2 mRNA vaccines encoding spike N-terminal and receptor binding domains

This publication in BioRxiv in 2022. This study used HEK293T cells to develop COVID-19 virus mRNA vaccines encoding spike gene N-terminal and RBD (receptor binding domain) domains.

Circular HER2 RNA positive triple negative breast cancer is sensitive to Pertuzumab

This research is published in Molecular Cancer in 2020. The study proposed that circular HER2 RNA expression in triple-negative breast cancer cells makes it sensitive to Pertuzumab drug treatment. Researchers used HEK293 cells for lentivirus production and transfection of circular HER2 gene in this study.

Antiviral role of IFITM3 in prototype foamy virus infection

This paper was published in the Virology Journal in 2022. This study utilized HEK293T cells to investigate the antiviral effect of IFITM3 (interferon-induced transmembrane protein 3) in prototype foamy virus (PFV) infection.

MiRNA-21 mediates the antiangiogenic activity of metformin through targeting PTEN and SMAD7 expression and PI3K/AKT pathway

This research paper in Nature Scientific Reports (2017) used HEK293T cells and studied that miRNA-21 mediates metformin-induced antiangiogenic effects by regulating PI3K/AKT signaling pathway and SMAD7 and PTEN gene expression.

MicroRNA-608 inhibits proliferation of bladder cancer via AKT/FOXO3a signaling pathway

This research is published in the Molecular Cancer journal in 2017. This study used HEK293 cells to investigate the antiproliferative potential of miRNA-608 against bladder cancer.

6. Resources for HEK293T cell line: Protocols, Videos, and More

The following are a few resources on HEK293T cells.

Transfecting HEK293T cells: This link will help you learn the transfection protocol for HEK293T cells.
HEK cell transfection: This video shows the general transfection protocol for the human embryonic kidney cell line.
Passaging cells: This video explains splitting or subculturing protocol for adherent cells.

Cell culture protocols

The cell culture protocols for HEK293T cells are listed here.

Splitting HEK293T cells: This site provides a detailed step-wise guide to subculture HEK293 cells.
Culturing human embryonic kidney cells: This link will provide you with the HEK293T cell culture protocol.

References

Tan, E., et al., HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors. Frontiers in Bioengineering and Biotechnology, 2021. 9.
Kim, M.J., et al., AMPKα1 Regulates Lung and Breast Cancer Progression by Regulating TLR4-Mediated TRAF6-BECN1 Signaling Axis. Cancers (Basel), 2020. 12(11).
Wang, Q., et al., Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2. Cell, 2020. 181(4): p. 894-904.e9.
Gale, E.C., et al., Hydrogel-based slow release of a receptor-binding domain subunit vaccine elicits neutralizing antibody responses against SARS-CoV-2. bioRxiv, 2021.
Kalsoom, A., et al., In Vitro Evaluation of Cytotoxic Potential of Caladium lindenii Extracts on Human Hepatocarcinoma HepG2 and Normal HEK293T Cell Lines. Biomed Res Int, 2022. 2022: p. 1279961.
Yu, Z., et al., LncRNA SNHG16 regulates trophoblast functions by the miR-218-5p/LASP1 axis. J Mol Histol, 2021. 52(5): p. 1021-1033.