Caco-2 Cell Bioassay: An In Vitro Method for Measuring Iron Bioavailability in Complex Food Sources

Weigh out the sample in a sterile 50-milliliter centrifuge tube. Adjust the pH using hydrochloric acid, and add 10 milliliters of physiological saline containing 140 millimolar sodium chloride and 5 millimolar potassium chloride. Then, set the gastric digestion process by adding 0.5 milliliters of the prepared porcine pepsin solution to the sample.

Next, incubate on a rocking shaker at a gentle setting, for 1 hour at 37 degrees Celsius, and initiate the intestinal digestion process of each sample by adjusting the pH to 5.5-6.0 with 1.0 molar sodium bicarbonate.

Add 2.5 milliliters of the pancreatic-bile solution to each sample tube, and adjust the pH to 6.9-7.0 with 1.0 molar sodium bicarbonate.

Using the solution containing 140 millimolar sodium chloride and 5 millimolar potassium chloride, add liquid so that each tube contains exactly 15 grams of total material.

Now, transfer 1.5 milliliters of each intestinal digest into the upper chamber of the well containing the Caco-2 cells of the 6-well culture plate. Replace the plate cover, and incubate at 37 degrees Celsius in 5% carbon dioxide on a rocking shaker at 6 oscillations per minute, for 2 hours.

Remove the insert ring with the digest. Then, add 1 milliliter of minimum essential medium at pH 7 to each well, and keep the plate back in the incubator for 22 hours.

Now, remove the cell culture medium and add 2 milliliters of 18 mega ohm (MΩ) water to the cell monolayer. Transfer it to a sonicator and harvest the entire cell lysate into microcentrifuge tubes for cell protein and cell ferritin analyses.