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HT-29 Cell Line: Colon Cancer Research and Experimental Therapeutics

HT-29 is a human colorectal cancer cell line with a wide range of cancer and toxicology research applications. Such as, these cells are sensitive to standard chemotherapeutic treatments against colorectal cancer. In addition, the HT-29 cell line is a well-characterized in vitro model to investigate the response of intestinal epithelium to bacterial infection. The HT-29 cells mimic the characteristics of mature intestinal cells, i.e., enterocytes, and thus are suitable for attachment and mechanistic studies.

This article will cover all aspects of the HT-29 cell line you need to know before working with it. Mainly it will include:

  1. HT-29 cells: Origin and general information
  2. Cell culture information of HT-29 cell line
  3. HT-29 cells: Advantages and Limitations
  4. HT-29 cells: Applications
  5. Research publications on HT-29 cells
  6. HT-29 cells Resources: Protocols, Videos & More

1.      HT-29 cells: Origin and general information

Before using any cell line, you should know about the general characteristics and origin of the cell line. This section will help you learn: What are the HT-29 cells? Is HT-29 a colorectal cancer cell line? What is the morphology of HT-29 cells?

What is the HT-29 cell line? HT-29 is a colorectal adenocarcinoma cell line established in 1964 by Trempe and Fogh. The cells were originally derived from the primary tumour of a Caucasian female (44 years old) with colorectal adenocarcinoma [1]. Since its establishment, this cell line has been used to study the biology of cancers.

Morphology of HT-29 cells: HT-29 cell line generally has epithelial-like morphology. Differentiated cells grow in monolayers, form tight cell-cell junctions, and possess an apical brush border. If these cells are grown in glucose deficient medium or given inducers like butyrate or acid, they exhibit enterocyte-like features.

Cell size: The diameter of the HT-29 colorectal cancer cell line is 11 μm.

Genome and ploidy: The modal number of chromosomes for the HT-29 cell line is 71. It may range from 68-72. Moreover, the streamlined chromosome number for HT-29 cells is hyper-triploid, with the 2S component occurring at the rate of 2.4 %.

What is HT29 MTX? HT29 MTX is a methotrexate-resistant cell line isolated from the original HT-29 human colon cancer cell line by adaptation to 10 µM Methotrexate. These cells present characteristics of mucous-secreting mature goblet cells.

Division and movement of colorectal cancer cells under the microscope.

2.      Cell culture information of HT-29 cell line

Knowing about the basic culture requirements of a cell line can make your work easy with it. You should know: what is the doubling time of the HT-29 cell line? What is the seeding density of HT-29 cells? What media for HT-29 cells?

Doubling time

The mean doubling time for the HT-29 cell line is 24 hours.


HT-29 is an adherent cell line. 

Seeding density

The seeding density recommended for HT-29 cells is 3 x 104 cells/cm2. Grown cells are washed with 1 X PBS and detached after incubation with Accutase for 8-10 minutes. Fresh media is added, and cells are centrifuged. The cell pellet is resuspended, and cells are dispensed into new flasks at recommended cell density.

Growth medium

Eagle's Minimum Essential Medium (EMEM) supplemented with 2.5 mM L-glutamine and 10 % FBS is used for optimal culturing of HT-29 cells. The medium should be replaced 2 to 3 times a week.

Growth conditions

HT-29 cells require a humified incubator with 37°C temperature and 5 % CO2 supply for growth.


These cells are stored in the vapour phase of liquid nitrogen at below -195°C.  


Freezing medium and process

The freezing medium used for the HT-29 cell line is CM-1 or CM-ACF. Cells are frozen by a slow freezing process that causes a 1 °C  drop in temperature and efficiently protects the viability of cells.

Thawing process

Frozen HT-29 cells are thawed in a water bath set at 37°C temperature. These cells can be resuspended and directly dispensed into the fresh growth medium flask. Contrary to this, the thawed cells can be centrifuged to remove freezing medium elements, resuspended in fresh medium, and cultured in flasks.

Biosafety level

Biosafety level 1 is used to handle and maintain the HT-29 cell line.

HT-29 cells under 10x and 20x magnification.

3.      HT-29  cells: Advantages and Limitations

Every cell line has unique pros and cons that distinguish it from other cell lines. This section will cover the main advantages and limitations associated with HT-29 cells.


The main advantages of HT-29 cells are:

Easy to Culture

HT-29 cell line is easy to handle and maintain, with no complex requirements for culturing. It is widely cultured in research laboratories.

In Vitro Model of Intestinal Cells

HT-29 cells can differentiate into mature intestinal cells (enterocytes and mucus-producing cells) under specific culture conditions. They are used as a cellular model to study food digestion and bioavailability.

High Transfection Efficiency

HT-29 colon cancer cells offer high transfection efficiency, making them suitable for gene expression and regulation studies. Both transient and stable transfection experiments can be performed using various methods.

Drug Sensitivity

HT-29 cells are sensitive to chemotherapeutic drugs such as fluorouracil and oxaliplatin, commonly used in colon cancer treatment. They are used to study drug resistance and screen potential therapeutic drugs for colorectal carcinoma.


The limitations of the HT-29 cell line are:


HT-29 cells, like other cancer cell lines, exhibit heterogeneity and may not form a uniform cell population. This can impact the reproducibility of results in certain assays and lead to minor variations in experimental outcomes.

Limited Differentiation

HT-29 cells have limited differentiation potential. They do not fully differentiate into intestinal or colon cells but exhibit only a few characteristic features. This limitation may restrict their use in studying more complex biological processes.

4.      HT-29  cells: Applications

HT-29 cell line is extensively cultured in research laboratories due to its wide range of applications. This section will cover only a few significant applications of HT-29 cells.

Cancer research: HT-29 is a colorectal adenocarcinoma cell line, so it is used to study cancer progression, development, and underlying molecular mechanisms. A study conducted in 2018 used the HT-29 cell line to investigate the cell molecules responsible for metastasis. This study observed that vimentin and CXCR4 genes regulate cell metastasis. Whereas transfecting microRNA-193a-5p mimic can reduce the mRNA levels of these genes and successfully decrease cancer cell metastasis [2].

Toxicology research: HT-29 cell line is extensively used in toxicology research. These colorectal carcinoma cells are used to assess the toxicity of different drug candidates using different assays. Such as, a study explored the toxicity potential of a plant Syzygium cumini ethanolic extract on the HT-29 cell line. The results showed significant suppression of HT-29 cell growth, indicating the promising anti-cancer properties of the extract [3].

Microbiology: HT-29 can be used as a host cell system to investigate the pathogenesis of potential microbes, i.e., bacteria and viruses. Researchers can infect HT-29 cells with microbes to investigate their virulence and host response. Such a study used HT-29 cells to explore lactic acid bacteria's probiotic properties [4]. Another study used HT-29 cells to evaluate the efficacy and safety of bacteriophage treatments [5].

5.      Research publications on HT-29 cells

This section will enlighten some notable publications on HT-29 cells that will help you devise your research study.

Evaluation of the Anti-cancer Effect of Syzygium cumini Ethanolic Extract on HT-29 Colorectal Cell Line

This study proposed Syzygium cumini ethanolic extract as a potential anti-cancer agent against colorectal cancer cell line HT-29. The extract significantly suppressed the growth of cancer cells.

Tetrandrine inhibits colon carcinoma HT-29 cells growth via the Bcl-2/Caspase 3/PARP pathway and G1/S phase

This publication in Bioscience Reports tested the anti-cancer activity of a compound, Tetradrine, on HT-29 cells. The results revealed that tetrandrine inhibits cancer cell proliferation and growth via regulating cell signalling pathways.

microRNA‐193a‐5p inhibits migration of human HT‐29 colon cancer cells via suppression of metastasis pathway

This study proposed that miRNA-193a-5p can potentially regulate HT-29 cell metastasis by regulating metastasis-related genes.

Bacteriophages are more virulent to bacteria with human cells than they are in bacterial culture; insights from HT-29 cells

This paper is published in Nature Scientific Reports in 2018. This study utilized HT-29 tumorigenic cell line as an intestinal cellular model to evaluate the efficacy of the proposed bacteriophage treatment.

Probiotic properties of lactobacilli and their ability to inhibit the adhesion of enteropathogenic bacteria to Caco-2 and HT-29 cells

In this study, researchers used HT-29 cells to investigate the probiotic activities of lactic acid bacteria. Interestingly, the study indicated that lactobacilli bacteria prevent pathogenic bacteria adhesion to colon cells.

6.      HT-29 cells Resources: Protocols, Videos & More

This section will cover some online resources available on HT-29 cells that will help learn their maintenance, culturing, and transfection protocols.

Cell culture protocols

The following resources will guide you about subculturing of HT-29 cells.

  • Culturing HT-29 cells: This link will provide basic knowledge about the growth medium, thawing, and subculturing of HT-29 cells.
  • Maintaining HT-29 cultures: This article contains all the useful information about HT-29 cell culturing and maintenance.

Transfection protocols

HT-29 cell line has great transfection efficiency, and it is frequently used in gene expression studies. The following resources describe the possible transfection protocols for these cells.

Videos related to HT-29 cell line

Here are some videos on this tumorigenic cell line culturing and maintenance.

We expect that the information in this article is valuable and sufficient for you to handle HT-29 cell line handling and maintenance. If you plan to use HT-29 cells in your study, order from us.


  1. Martínez-Maqueda, D., B. Miralles, and I. Recio, HT29 cell line. The Impact of Food Bioactives on Health: in vitro and ex vivo models, 2015: p. 113-124.
  2. Shirafkan, N., et al., microRNA‐193a‐5p inhibits migration of human HT‐29 colon cancer cells via suppression of metastasis pathway. Journal of cellular biochemistry, 2019. 120(5): p. 8775-8783.
  3. Khodavirdipour, A., R. Zarean, and R. Safaralizadeh, Evaluation of the anti-cancer effect of Syzygium cumini ethanolic extract on HT-29 colorectal cell line. Journal of gastrointestinal cancer, 2021. 52: p. 575-581.
  4. Fonseca, H.C., et al., Probiotic properties of lactobacilli and their ability to inhibit the adhesion of enteropathogenic bacteria to Caco-2 and HT-29 cells. Probiotics and antimicrobial proteins, 2021. 13: p. 102-112.
  5. Shan, J., et al., Bacteriophages are more virulent to bacteria with human cells than they are in bacterial culture; insights from HT-29 cells. Scientific reports, 2018. 8(1): p. 5091.


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