The intestinal barrier must efficiently absorb nutrients while simultaneously blocking harmful substances, a dual function that is challenging to study in living systems. To overcome this complexity, scientists use cell lines, which are cells grown indefinitely in a laboratory setting, to create simplified tissue models. The Caco-2 cell line is the most widely used model for mimicking the intestinal epithelium. This standardized cell culture system provides a high-throughput tool for understanding how substances, from pharmaceutical compounds to dietary components, are absorbed across the gut wall.
Defining the Caco-2 Model
The Caco-2 cell line was derived from a human colorectal adenocarcinoma (colon cancer) in 1977. Despite its cancerous origin, this cell line spontaneously differentiates when grown in culture. This process causes the cells to morphologically and functionally resemble enterocytes, the absorptive cells lining the small intestine.
As Caco-2 cells grow and divide, they develop a phenotype characteristic of small intestinal cells. This includes forming a dense brush border, a layer of microscopic microvilli on the cell surface. The differentiated cells also express small intestine-specific enzymes, such as disaccharidases and peptidases, which break down sugars and proteins. This spontaneous maturation into enterocyte-like cells makes the Caco-2 line a relevant model for intestinal function.
Forming the Intestinal Barrier Monolayer
The primary utility of the Caco-2 cell line is its capacity to form a polarized monolayer that mimics the intestinal barrier. Polarization involves developing two distinct sides: the apical and basolateral surfaces. The apical side, featuring microvilli, faces the culture medium and represents the intestinal lumen where food and drugs reside.
The basolateral surface rests on the culture insert and represents the side facing the body’s circulation, where absorbed compounds enter the bloodstream. Tight junctions, formed by protein complexes between neighboring cells, act as a seal. These junctions dictate the movement of substances between cells, a route known as paracellular transport.
The integrity of this barrier is quantified by measuring the Trans-Epithelial Electrical Resistance (TEER). TEER involves passing a small electrical current across the monolayer and measuring the electrical resistance, which is inversely related to permeability. A high TEER value (typically 150 to over 400 \(Omega cdot text{cm}^2\)) confirms that the tight junctions have sealed the space between the cells. TEER provides a non-invasive metric for assessing if the monolayer is sufficiently developed for transport experiments.
Essential Uses in Drug and Nutrient Research
The Caco-2 monolayer assay is recognized as the standard for predicting the absorption of orally administered drugs in the human small intestine. Pharmaceutical companies use this model extensively to determine a compound’s apparent permeability coefficient (\(P_{text{app}}\)), which measures how quickly a substance moves from the apical (gut) side to the basolateral (blood) side. Correlating \(P_{text{app}}\) values with known human absorption rates allows researchers to predict the bioavailability of new drug candidates early in development.
The model allows for the study of both passive diffusion and active transport mechanisms. By measuring transport in both directions (apical-to-basolateral and basolateral-to-apical), scientists can identify compounds that are substrates for efflux pumps, such as P-glycoprotein. These pumps actively push drugs back into the gut lumen, meaning a high basolateral-to-apical transport rate indicates poor absorption in vivo.
The Caco-2 system is also a powerful screening tool in nutritional science and toxicology. Researchers investigate how vitamins, minerals, and bioactive food components are absorbed, or assess the safety of food additives and contaminants. For example, the model predicts iron bioavailability by measuring the formation of the iron-storage protein ferritin within the cells, which correlates strongly with human trial data. The monolayer is also used in toxicology studies to evaluate cytotoxicity and drug safety following exposure to xenobiotics.
Understanding the Model’s Scientific Limitations
Despite its widespread use, the Caco-2 model is a simplification with several biological limitations. The most significant drawback is the absence of a functional mucus layer. In the human gut, mucus forms a thick, protective gel that compounds must cross before reaching the enterocytes. Since Caco-2 cells do not secrete sufficient mucus, the model can lead to an overestimation of the absorption of some compounds.
Addressing the mucus limitation often requires scientists to use co-culture systems, combining Caco-2 cells with a mucus-secreting cell line, such as HT29-MTX.
An additional issue is the difference in protein expression, as the Caco-2 line is derived from the colon, not the small intestine. This results in the under- or over-expression of specific drug transporters and metabolic enzymes, such as a deficiency of Cytochrome P450 3A4 (CYP3A4). Furthermore, the tight junctions in the Caco-2 monolayer are often tighter than those in the human small intestine. This can lead to an under-prediction of paracellular transport for small, hydrophilic molecules that typically pass between cells.