Critical Suitability\nEvaluation of Caco‑2 Cells\nfor Gut-on-a-Chip

Currently, culturing Caco-2 cells in a Gut-on-a-chip\n(GOC) is well-accepted\nfor developing intestinal disease models and drug screening. However,\nCaco-2 cells were found to overexpress surface proteins (e.g., P-gp)\ncompared with the normal intestinal epithelial cells <i>in vivo</i>. To critically evaluate the challenge and suitability of Caco-2\ncells, a GOC integrated with a carcinoembryonic antigen (CEA) biosensor\nwas developed. This three-electrode system electrochemical sensor\ndetects CEA by antigen–antibody specific binding, and it exhibits\nhigh selectivity, excellent stability, and good reproducibility. Under\ndynamic culturing in the GOC, Caco-2 cells exhibited an intestinal\nvillus-like structure and maintained tissue barrier integrity. Meanwhile,\nCEA was discovered to be secreted from 0 to 0.22 ng/mL during the\n10-day culturing of Caco-2 cells. Especially, CEA secretion increased\nsignificantly with the differentiation of Caco-2 cells after 6 days\nof culturing. The sustained high-level CEA secretion may induce cells\nto avoid apoptotic stimuli, which faithfully reflects the efficacy\nof a new drug and the mechanism of intestinal disease. Different kinds\nof cell types (e.g., intestinal primary cells, stem cell-induced differentiation)\nin the GOC should be attempted for drug screening in the future.