Unraveling Caco-2 cells through functional and transcriptomic assessments

The static Caco-2 monolayer is an extensively utilized model for predicting the permeability of small molecules during the drug development process. While these cells can differentiate and develop key functional and morphological features that emulate human enterocytes, they do not fully replicate the complexity of human intestinal physiology. In this study, we investigated functional and morphological aspects of Caco-2 cells, alongside their transcriptomic profiles, with a particular emphasis on genes encoding drug-metabolizing enzymes and drug transporters. We found that Caco-2 cells not only established a robust and bio-relevant permeable intestinal barrier but also demonstrated functional maturity and differentiation in the intestinal epithelium, substantiated by the activities of important enzymes and an efflux transporter. However, our targeted gene expression analyses revealed that substantial disparities were found in mRNA transcript levels among Caco-2 cells and human biopsy samples. These findings highlight that, although Caco-2 cells are valuable for assessing the passive transport of drugs, their accuracy for predicting active transport or small intestinal drug metabolism is constrained by their transcriptomic divergence from human intestinal tissues. This study highlights the importance of understanding the Caco-2 model’s inherent limitations and provides insights that could inform its appropriate application in drug development and regulatory decision-making. • Comprehensive evaluation of Caco-2 monolayers demonstrates their ability to form a robust and tight intestinal barrier with active efflux transporters and metabolizing enzymes. • Targeted gene expression profiling reveals significant disparities in drug-metabolizing enzymes and drug transporters between Caco-2 cells and human intestinal tissues. • Findings underscore the limitations of static Caco-2 monolayers, including insufficient expression of key drug-metabolizing enzymes and transporters, which may constrain their accuracy in predicting drug absorption, first-pass metabolism, and transporter-mediated interactions in human intestinal physiology.