HCT116 cells

HCT116 are human colon carcinoma cells. They are extensively used in biomedical studies involving cancer research and drug screening. In addition, the HCT116 cell line has wide-ranging applications in tumorigenicity studies.

This article contains all the useful information about the HCT116 cell line you need to know before working with it. Mainly, it will include the following: 

  1. General characteristics and origin of HCT116 cells
  2. Culturing information of HCT116 cell line
  3. HCT116 cell line: Advantages & Limitations
  4. Research applications of HCT116 cell line
  5. HCT116 cells: Research publications
  6. Resources for HCT116 cell line: Protocols, Videos, and More

 1.      General characteristics and origin of HCT116 cells

Before culturing a cell line, the first thing that comes to mind is its origin and general characteristics like morphology, cell size, etc. This section will cover basic information about the HCT116 cell line. You will learn What is HCT116 cell line? What are HCT116 cell line characteristics? What is the diameter of HCT116? How many chromosomes are in HCT116?

  • The human colorectal carcinoma cell line, HCT116, is isolated from the colon of a 48-year-old Caucasian male with colon cancer. HCT116 cells are known for codon 13 (G13D) mutation in the KRAS gene, a proto-oncogene of the RAS/RAF/MEK/ERK cascade. This HCT116 mutation is associated with tumorigenesis of the cell line.
  • The colon cancer cell line HCT116 possesses an epithelial cell-like morphology.
  • HCT116 cells grow as monolayers and form larger spheres with a diameter ranging from 150-400 µm.
  • HCT116 cells possess a near-diploid set of chromosomes. Around 70% of cells consist of 45 chromosomes. However, the overrepresentation of chromosomes 8, 10, 16, and 17 occurs on the long arms. HCT116 cells lack chromosome Y.

HCT116 Vs HT 29

Both HCT116 and HT 29 cells represent human colorectal carcinoma. Herein HCT116 is a highly aggressive cancer cell line and has no or little differentiation capacity, whereas HT29 can differentiate into enterocytes and mucin-expressing cell lineages.

Pre-cancerous polyps growing in the colon.

2.      Culturing information of HCT116 cell line

HCT116 cancer cell line has extensive research applications. Before culturing HCT116 cancer cells, you need to learn the following key points. You should know: What is HCT116 media? What is the doubling time of HCT116 cells? What is HCT116 cell seeding density? How do you thaw HCT116 cells?

Key Points for Culturing HCT116 Cells

Doubling Time:

The doubling time of HCT116 cancer cells is around 25 to 35 hours.

Adherent or in Suspension:

HCT116 colon cancer cell line is adherent, and cells grow in monolayers.

Seeding Density:

A cell seeding density of 2 x 104 cells/cm2 is recommended for the HCT116 cancer cell line. For seeding, grown cells are detached with accutase solution after washing with 1x PBS. Cells are resuspended in a medium and centrifuged. Recovered cells are carefully resuspended and poured into a new flask.

Growth Medium:

McCoys 5a is recommended as HCT116 cell culture media. This media is supplemented with 3.0 g/L L-glucose, 1.5 mM L-glutamine, 3.0 g/L NaHCO3, and 10% fetal bovine serum for optimum cell growth. Media should be renewed 1 to 2 times per week.  

Growth Conditions (Temperature, CO2):

HCT116 cells are kept in a humidified incubator set at 37°C temperature and with a 5% CO2 supply.


HCT116 cells can be stored in an electric freezer at below -150°C temperature and the vapor phase of liquid nitrogen.

Freezing Process and Medium:

CM-1 or CM-ACF medium is used to freeze the HCT116 cell line. The slow freezing method opted for freezing cells. This permits a gradual 1°C drop in temperature, thus protecting cell viability.

Thawing Process:

HCT116 cells are thawed at 37°C temperature in the water bath. Cells are added with media and centrifuged to completely remove freezing media. The cell pellet is resuspended in a fresh culture medium, and cells are dispensed in ne flask for culturing.

Biosafety Level:

HCT116 cell culture is maintained in the biosafety level 1 laboratory.

Semi-confluent HCT116 cells magnified 20x and 10x.

3.      HCT116 cell line: Advantages & Limitations

This section of the article will go over the advantages and limitations associated with the HCT116 cancer cell line.


The main advantages of the HCT116 cell line are:

Colorectal Cancer Model:

Widely used in vitro model for studying colon cancer, the third most prevalent cancer worldwide.


Approximately 70% of HCT116 cells exhibit similar genetic attributes, providing a homogeneous culture. This characteristic is advantageous for gene expression, cell signaling, and drug screening and response studies.

Transfection Amenability:

HCT116 cells demonstrate excellent transfection flexibility, particularly with viral vectors. This attribute makes them highly suitable for gene therapy research.


The limitation of HCT116 cells is:

Susceptibility to culture conditions:

HCT116 cells are sensitive to changes in temperature, pH, and nutrient availability, which can impact their response to experimental treatments and compromise result reproducibility.


4.      Research applications of HCT116 cell line

HCT116 cell line has a wide range of applications in cancer research. Some prominent applications are:

Cancer biology

HCT116 colon cancer cell line is used to study colon cancer progression and development. Moreover, it helps enlighten underlying mechanisms and signaling pathways involved in cancer proliferation, migration, and invasion. A study used HCT116 cells to study genes involved in drug resistance development. Researchers overexpressed the MDR1 gene in colon cancer cells and observed the expression of NOX (NADPH oxide) isoforms and Nrf2. The study revealed that NOX2 and Nrf2 upregulation causes chemoresistance in cancer cells; thus these genes can be targeted to overcome resistance development during cancer therapy [1].

Likewise, research conducted in 2021, reported that NF-κB Signaling Pathway is involved in the regulation of colon cancer proliferation and migration. Thus it can be targeted to develop new and effective therapeutics against colorectal carcinoma [2].

Toxicology/drug development

HCT116 cell line is applied as a screening model for new cancer drugs. Several studies have been conducted to assess the efficacy and toxicity of anticancer drugs, including natural products and chemically synthesized nanoparticles. Such as, the research assessed the cytotoxicity of synthesized silver nanoparticles from the extracts of a herbal drug, Caesalpinia pulcherrima in HCT116 cells [3].  

In a study, researchers have used the HCT116 cancer cell line to evaluate the anticancer potential of Cocoa tea water extract. They found that Cocoa tea extract reduces colon cancer proliferation and induces cell death [4].

Another study used HCT116 cancer cells and discovered that air potato, Dioscorea bulbifera, extracts exhibit pro-apoptotic activity in colorectal carcinoma cells via activation of the JNK signaling cascade and suppression of ERK1/2 gene [5].

5.      HCT116 cells: Research publications

This section will go over a few significant and most cited recent publications featuring the HCT116 cell line.

Cytotoxicity study of Piper nigrum seed mediated synthesized SnO2 nanoparticlestowards colorectal (HCT116) and lung cancer (A549) cell lines

This study was published in the Journal of Photochemistry and Photobiology B: Biology (2017). Researchers used HCT116 colon cancer and A549 lung cancer cell lines to evaluate the cytotoxic effects of piper nigrum seed-mediated synthesized tin oxide nanoparticles.

Long non-coding RNA SNHG15 interacts with and stabilizes transcription factor Slug and promotes colon cancer progression

This research in Cancer Letters (2018) proposed that lncRNA SNHG15 promotes colon cancer cell migration in colorectal cancer cell lines, including HCT116.

Overexpression of long non-coding RNA TUG1 promotes colon cancer progression

This paper was published in the Medical science monitor journal in 2016. The study found that oncogenic LncRNA TUG1 promotes the proliferation and migration of HCT116 colon cancer cells.

Drug resistance induces the upregulation of H2S-producing enzymes in HCT116 colon cancer cells

This research in Biochemical Pharmacology journal (2018) proposed that drug resistance development upsurges the levels of H2S-producing enzymes in HCT116 colon cancer cells.

Apoptotic and antiproliferative effects of Inula viscosa L. water extract in the expression of microRnas on HCT 116 cell line: an in vitro study

This research paper in the International Journal of Environmental Health Research (2023) proposed that Inula viscosa L. extract exerts an anticancer effect on HCT116 colorectal cancer cells via regulating microRNAs.

6.      Resources for HCT116 cell line: Protocols, Videos, and More

The following are a few resources on HCT116 cells.


  1. Waghela, B.N., F.U. Vaidya, and C. Pathak, Upregulation of NOX-2 and Nrf-2 promotes 5-fluorouracil resistance of human colon carcinoma (HCT-116) cells. Biochemistry (Moscow), 2021. 86: p. 262-274.
  2. Yang, M., et al., Astragalin inhibits the proliferation and migration of human colon cancer HCT116 cells by regulating the NF-κB signaling pathway. Frontiers in Pharmacology, 2021. 12: p. 639256.
  3. Deepika, S., C.I. Selvaraj, and S.M. Roopan, Screening bioactivities of Caesalpinia pulcherrima L. swartz and cytotoxicity of extract synthesized silver nanoparticles on HCT116 cell line. Materials Science and Engineering: C, 2020. 106: p. 110279.
  4. Gao, X., et al., Cocoa tea (Camellia ptilophylla) induces mitochondria-dependent apoptosis in HCT116 cells via ROS generation and PI3K/Akt signaling pathway. Food Research International, 2020. 129: p. 108854.
  5. Hidayat, A.F.A., et al., Dioscorea bulbifera induced apoptosis through inhibition of ERK 1/2 and activation of JNK signaling pathways in HCT116 human colorectal carcinoma cells. Biomedicine & Pharmacotherapy, 2018. 104: p. 806-816.