The Influence of Culture Time and Passage Number on the Morphological and Physiological Development of Caco-2 Cells

Abstract The Caco-2 cell line is used by many investigators as a model of the intestinal epithelium to study nutrient uptake and transport. Our goal was to create an awareness of inherent variabilities in the Caco-2 cell line which may influence their suitability as a model or their application to specific problems. To study the influence of passage on the model, cultures were monitored from passage 20 to 109. Transepithelial electrical resistance (TEER) and sucrase activity (measured in 21-day-old cultures) increased through about passage 36. TEER values declined after about passage 60; sucrase remained elevated but variable. Cells at passage 22, 33, and 72 were grown simultaneously for 24 days. Older-passaged cells reached plateau phase sooner. Before Day 15, passage 72 cells had higher TEER and lower permeability to 14C-mannitol than passages 22 and 33; however, after Day 15 all passages showed similar permeability. On Day 21, passage 72 cells had significantly lower alkaline phosphatase activity than did the other passages. Electron microscopy did not reveal any major morphological differences between the passages; however, it did show that some areas of cells grown on membranes were not monolayers but were several cells thick with varied morphology. Investigation of the formation of these multilayered areas showed them to be an inherent part of cell growth under the conditions used. These results emphasize the inherent variability in Caco-2 cell models and emphasize the need to monitor closely the culture characteristics during growth and differentiation under specific experimental conditions.