In vitro models can be useful tools in developing objectives that warrant testing in animal and human subjects. For nutrient absorption studies, Caco-2 cell monolayers have been widely used for defining certain aspects of nutrient uptake and transfer across epithelial cells. For a practical example, Caco-2 cell monolayers have been applied as a screening tool to address the food factors associated with iron deficiency anemia (IDA). IDA is the most common micronutrient deficiency worldwide and

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... primarily due to poor dietary Fe bioavailabiity. Development of foods with improved iron bioavailability is essential to alleviating this critical deficiency. Human and animal studies of iron absorption from food are time-consuming, expensive and offer limited capacity to address luminal interactions of iron and food ingredients. These factors have led scientists to pursue rapid, high-throughput in vitro methods that enable analysis of food-iron interactions and prediction of Fe bioavailability for humans. As a result, a simulation of in vitro digestion combined with Caco-2 cell monolayers was developed. The primary reason for this evolution of methodology is the fact that Fe solubility does not always correlate with Fe availability. Addition of the Caco-2 cell monolayer adds a living component to the model system that in theory should reflect the key step in iron bioavailability (i.e. uptake of iron by the enterocyte). In vitro digestion and Caco-2 cell culture conditions can vary significantly, and if proper physiological and cell culture principles are not applied, then the in vitro method can generate results that are not a true reflection of in vivo effects. Therefore, it is critical that an in vitro model be assessed under a broad range of conditions and validated against known effects in humans. This review summarizes the development, validation, and application of currently the most broadly applied in vitro digestionlCaco-2 cell culture model, providing it as an example of how epithelial cell lines can be used in practical nutrition research.