Caco-2 cells

The Caco-2 cell line is a widely-used model of the intestinal epithelial barrier in scientific research. These cells were initially derived from colon carcinoma in a 72-year-old Caucasian male using the explant culture method. One of the remarkable features of Caco-2 cells is their ability to differentiate into a single layer of cells that resemble absorptive enterocytes with a brush border layer found in the small intestine. However, Caco-2 cells are heterogeneous and display various properties.

Drug absorption and metabolism primarily focus on the fully differentiated villus cells within the intestinal epithelium. A valuable tool for studying drug and nutrient transport and metabolism would be an in vitro cell culture system consisting of a viable, polarized, and fully differentiated monolayer of villus cells similar to those found in the small intestine.

Medically accurate 3d animation of the intestinal villi

The Caco-2 cell line exhibits a well-differentiated brush border and tight junctions. It expresses typical small-intestinal microvillus hydrolases and nutrient transporters, making it a widely popular in vitro model for studying drug and nutrient transport and metabolism. It is used to rapidly assess the cellular permeability of drug candidates, elucidate drug transport pathways, assess formulation strategies, determine optimal physicochemical characteristics for drug diffusion, and evaluate the potentially toxic effects of drug candidates or formulation components on this biological barrier.

  1. What Makes the Caco-2 Cell Line Unique?
  2. Applications of the Caco-2 Cell Line
  3. Five Advantages of the Caco-2 Cell Line
  4. Instructions for Handling and Culturing Caco-2 Cells

1.        What Makes the Caco-2 Cell Line Unique?

The Caco-2 cell line has distinct characteristics that set it apart from other cell types. These include:

  • In culture, Caco-2 cells form a cylindrically polarized monolayer with brush border enzyme-secreting microvilli developing on their apical side and uniform tight junctions between adjacent cells.
  • Upon reaching confluence, the unidirectional flux of ions and water through the polarized monolayer leads to the formation of domes in the cultures.
  • Caco-2 cells express markers of colonocytes.
  • Late passage cells grow in multilayers, affecting TFER (transepithelial electric resistance) measurements.
  • The Caco-2 culture is heterogeneous and contains subpopulations with differing morphologies.

2.       Applications of the Caco-2 Cell Line

The Caco-2 cell line is a critical component of the drug discovery process, serving as the principal in vitro cellular model for drug absorption and permeability in the intestinal epithelium. Additionally, the Caco-2 cell line is widely used to study drug transport mechanisms due to functional brush border enzymes and transport proteins. Here are some of the various applications of the Caco-2 cell line:

Drug delivery at the intestinal cell level

  • Bioactive Food Components: The Caco-2 cell line can investigate the effects of microbiota, microbial metabolites, food digest, and bioactive components on the intestinal epithelium's barrier function and underlying processes.
  • Analyze the Intestinal Epithelium: Researchers use the Caco-2 cell line to determine how medications or dietary components traverse the intestinal epithelium (paracellular vs. transcellular, passive vs. carrier-mediated).
  • Mucosal Toxicity: The Caco-2 cell model is useful when investigating the mucosal toxicity of medication candidates or dietary metabolites.
  • Transport of Bioactive Compounds: The Caco-2 cell line helps investigate the effects of different formulation components (such as adjuvants or food matrices) on the transport of bioactive compounds through the intestinal epithelial membrane. It can also identify bioactive molecules with suitable physiochemical characteristics for transcellular or paracellular passive diffusion through the intestinal epithelium and investigate the interactions between bioactive compounds during transport.
  • Investigation of Efflux Systems: The Caco-2 cell line can be used to investigate the function and molecular basis of intestinal epithelial efflux systems, including multi-drug resistance proteins like P-glycoprotein.

Fluorescence microscopy of Caco2 monolayers labeled with a ZO-1 specic antibody. ZO-1, Tight junction protein-1, is a peripheral membrane protein encoded by the TJP1 gene in humans and has a molecular weight of 220 kD. ZO-1 is a member of the family of zonula occludens proteins and is associated with tight junctions. ZO-1 is a scaffold protein that cross-links and anchors tight junction strand proteins, fibril-like structures in the lipid bilayer, to the actin cytoskeleton. The protein is located on the cytoplasmic membrane surface of intercellular tight junctions and is believed to be involved in signal transduction at cell-cell intersections. The TJP1 gene has been found to encode two distinct isoforms of ZO-1, each with different functions.

3.       Five Advantages of the Caco-2 Cell Line

While it is challenging to list all the potential benefits of the Caco-2 cell line, here are some of its advantages:

  1. Fast Differentiation: Caco-2 cells differentiate rapidly to express mature small intestinal enterocytes' morphological and functional properties.
  2. High TEER Values: The polarized Caco-2 cell layer exhibits TEER (transepithelial electrical resistance) values that are four times higher than those of HT29 monolayers, making them a valuable tool for studying epithelial barrier function.
  3. Cholesterol Transport: The Caco-2 cell line is an excellent model for studying how cholesterol moves through the body and the expression of cholesterol transporters.
  4. Expression of Receptors and Enzymes: Caco-2 cells express most receptors, transporters, and drug-metabolizing enzymes found in normal epithelium, such as aminopeptidase, esterase, and sulfatase.
  5. Lack of P-450 Enzyme Activity: Notably, the Caco-2 cell line does not exhibit P-450 metabolizing enzyme activity, which is useful when studying drug metabolism pathways that do not involve this enzyme family.

Caco-2 cells at 20x and 10x magnification

Limitations of the Caco-2 Cell Model

While the Caco-2 cell model is a valuable tool for investigating intestinal epithelial features, it has several limitations when compared to normal intestinal epithelium:

  • Multiple Cell Types: Normal human epithelium contains more than one cell type, not only enterocytes, whereas the Caco-2 cell line only contains enterocytes.
  • Absence of Mucus and Unstirred Water Layer: When using the Caco-2 cell line, mucus and the unstirred water layer near the epithelium is absent.
  • Non-Cellular Parameters: Several non-cellular parameters, such as bile acids and phospholipids, will affect the absorption of a particular compound in cells. In vivo, compound solubility in the mucus layer plays a role in absorption, and the unstirred water layer near the epithelium will significantly impact uptake.

Related Cell Lines to Caco-2 Cells

All cell lines mentioned below are used as in vitro models of the intestinal epithelial barrier and have diverse characteristics and applications in research.

Cell Line


Characteristics and Applications


Human ileocecal adenocarcinoma cells

Similar to Caco-2 cells and used in toxicological and cancer research


Rat small intestine epithelial cells

Typical in vitro model of the intestinal epithelial barrier and essential for digestion, nutrition absorption, and defense against microbial infections


Epithelial-like cells isolated from a primary colon tumor of a 44-year-old female patient with colon adenocarcinoma

Useful for studies in oncology and toxicity and may serve as a transfection host


Mucous-secreting cell line derived from HT29 cells

Forms tight junctions and produces mucus, similar to gastric cells and Caco-2 cells


HT29 subclones differentiated into mature goblets with methotrexate

Useful for studying the differentiation and maturation of goblet cells in the colon

4.       Instructions for Handling and Culturing Caco-2 Cells

To culture and maintain Caco-2 cells, follow these steps:

  1. Remove the culture media and wash the adhering cells with phosphate-buffered saline (PBS) without calcium and magnesium ions (3-5 ml PBS for T25 and 5-10 ml for T75 cell culture flasks).
  2. Completely cover the cell sheet with Accutase (1-2 ml per T25, 2.5 ml per T75 cell culture flask) and leave it at room temperature for 8-10 minutes.
  3. Reconstitute the cells in fresh media (10 ml), centrifuge for 3 minutes at 300 g, and carefully transfer the cells to new flasks.
  4. For recovery from the freezing procedure, allow the cells at a density of 5 x 104 cells/cm2 to stick to the plate for at least 24 hours after thawing.
  5. The doubling time for Caco-2 cells is 60-70 hours, and the recommended split ratio is 1:2 to 1:3. 90 percent monolayer confluence is reached at 1 x 104 cells/cm2 after four days.
  6. Replace the medium for confluent cultures every two to three days or less frequently if they are not sub-cultured.
  7. Tight junction expression in Caco-2 cells is typically evaluated by measuring trans-epithelial electrical resistance (TEER).
  8. For drug discovery experiments, cells may be cultivated on semi-permeable supports.

Final Thoughts

In conclusion, several critical parameters must be controlled to provide reliable and authentic information using Caco-2 cell monolayer systems. It is essential to validate the integrity of monolayers before conducting transport studies with Caco-2 cells and to avoid cell culture contamination. We have provided general protocols for handling the Caco-2 cell line. Still, it is essential to note that the standard procedure requires 21 days of culture to obtain a polarized monolayer with the full expression of intercellular contacts and tight junctions. We hope this information has covered all essential aspects of the Caco-2 cell line and helped to provide a comprehensive understanding of these cells.

For those seeking to purchase the Caco-2 cell line for experimentation and who want to ensure authenticity and quality, consider buying from us!