The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Knowledge of iron bioavailability is essential for the assessment of the nutritional quality of iron in foods. The Caco-2 cell bioassay was developed to meet this critical research need. This bioassay is a cost effective and high throughput approach for determining iron bioavailability from different diets.

It can be used to characterize factors influencing iron bioavailability, and refining in vivo study objectives. Demonstrating the procedure will be Yongpei Chang, a research technician from my laboratory. To begin, culture the Caco-2 cells for 7 to 10 days, and once sufficient cells are available seed the cells in a non collagen coated flask.

Then grow the cells in flasks for seven days and change the medium on an alternate day. On the seventh day use the cells for seeding the multi-well plates. Seed the Caco-2 cells at a density of 50, 000 cells per centimeter square in six well collagen coated plates.

Add DMEM supplemented with HEPES, FBS and 1%antibiotic antimycotic solution to the plates. Then incubate for 12 days at 37 degrees Celsius with 5%carbon dioxide. Keep changing the medium at least every two days on a consistent daily schedule.

Then prepare minimum essential medium supplemented with pipes, antibiotic antimycotic solution, hydrocortisone, insulin, selenium, triiodothyronine and epidermal growth factor. Replace the culture medium with two milliliters of this prepared medium. On the next day, replace it with one milliliter of the minimum essential medium at pH 7.

Now create a sterilized insert ring using a silicone O-ring fitted with an acid washed dialysis membrane. On the 13th day, remove the inserts from the refrigerator, drain and replace the water with 0.5 molar hydrochloric acid. Leave it in a laminar flow hood for at least one hour prior to use.

Then drain 0.5 molar hydrochloric acid and rinse with sterile 18 megaohm water. Store in sterile 18 megaohm water in a laminar flow hood until ready to use. Create a two chamber system by inserting a ring into the wells containing the cells of the six well plate and then return the plate with the inserts to the incubator.

For preparing the pancreatin-bile solution, add 87.5 grams of weak cation exchange resin to the solubilized pancreatin and bile extract and use a shaker for 30 minutes at room temperature to mix the components. Pour the slurry into a large column and drain the column. Then centrifuge the column eluent.

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 five 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 one hour at 37 degrees Celsius and initiate the intestinal digestion process of each sample by adjusting the pH to 5.5 to 6.0 with 1.0 molar sodium bicarbonate. Add 2.5 milliliters of the pancreatin bile solution to each sample tube and adjust the pH to 6.9 to 7.0 with 1.0 molar sodium bicarbonate. Using the solution containing 140 millimolar sodium chloride and five millimolar potassium chloride, add liquids 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 six well culture plate. Replace the plate cover and incubate at 37 degrees Celsius in 5%carbon dioxide on a rocking shaker at six oscillations per minute for two hours. Remove the insert ring with the digest.

Then add one 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 two milliliters of 18 megaohm water to the cell mono layer. Transfer it to a sonicator and harvest the entire cell lysate into micro centrifuge tubes for cell protein and cell ferritin analyses.

The manteca varieties showed higher iron availability relative to reference controls of the white and red modeled color classes for two consecutive harvest years. The iron bioavailability from white and yellow bean varieties showed an increase after processing them into flour. However, iron bioavailability showed a decrease in the cranberry, red kidney and black varieties.

Proper Caco-2 cell monolayer culturing is essential for this bioassay's successful use. Precise attention to the details of the growth and maintenance is key to the consistent response of the bioassay. This model negates the disadvantages and high cost of other approaches and enables researchers to identify mechanisms and more fully evaluate factors that inhibit and promote iron bioavailability.