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

NOTE: As a convenient point of reference for readers, the following methodology describes the specific culture conditions and materials required for the measurement of Fe bioavailability from 20 experimental samples, plus the required quality controls, in a run of the bioassay. Increasing the number of samples beyond this capacity is not recommended due to the time required for various cell culture and in vitro digestion steps within the bioassay.

1. Choosing the amount of samples

1. For solid or liquid foods, determine an amount of food that can be considered representative of the sample to be tested.
   1. In testing for Fe bioavailability from a bean variety, use 100-150 g of bean seed and process this amount to a homogeneous sample.
   2. For liquid samples such as fortified juices, milk products, and sports beverages, ensure that the food is well mixed prior to sampling.
      NOTE: The amount of bean seed material mentioned above is essential to account for the inherent differences between seeds of this staple crop.

2. Preparation of samples

1. Rinse off soil and dust from any food sample with distilled-deionized water before processing.
2. Process the appropriate amount of sample as per the experimental objectives, such as by cooking method and milling.
   NOTE: For cooking and processing, it is critical to use cookware and equipment that is not a potential source of contaminant Fe. Stainless steel equipment does not contaminate; however, equipment such as stone mill grinders, cast iron cookware, and any non-stainless steel equipment containing Fe can add significant amounts of contaminant Fe. A standard stainless steel coffee grinder is often adequate for grinding.
3. Lyophilize and grind to a homogeneous powder.
   NOTE: Once homogenized, research has shown that three independent replications of analysis are adequate for each food being measured.
   1. If sample homogeneity is difficult to achieve, revise the formulation or processing of the product. If this is not possible, add replications if the non-homogeneity is not severe.
   2. For most homogeneous solid foods, use 0.5 g of lyophilized sample per replicate. If necessary, use up to 1.0 g of sample per replicate, but check if more than 0.5 g yields a benefit in the degree of response.
      NOTE: Amounts higher than 0.5 g of solid foods may clog the dialysis membrane (see below).
   3. Use 1-2 mL of liquid samples.
      NOTE: Lyophilization is often not necessary for liquid samples.

3. Caco-2 cell culture

1. Stock cultures
   1. Acquire Caco-2 cells from a certified supplier.
   2. Culture the cells from stock vials at 37 °C in an incubator with a 5% CO₂ air atmosphere (constant humidity) using Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 25 mM HEPES (pH 7.2), 10% (v/v) fetal bovine serum (FBS), and 1% antibiotic-antimycotic solution.
   3. Once sufficient cells are available, usually after 7-10 days of culture, seed the cells in non-collagen-coated flasks at a density of 30,000 cells/cm².
   4. Choose the flask size depending on the number of cells available and needed for seeding multiwell plates.
      NOTE: In general, the T225 (225 cm²) flasks work best for experiments where 11 multiwell (6-well; 9.66 cm²/well) plates are used (10 plates for sample comparisons, 1 plate for quality controls) per bioassay.
   5. Grow cells in flasks for 7 days, changing the medium every other day, and use on the 7th day for seeding the multiwell plates.
      NOTE: It is recommended to use a passage range of no more than 10-15 passages from when the cells are started from stock culture and subsequently used in a series of experiments. Cell culture passages should be limited as a broad range of passages can result in adaptive changes in the cell line and, thus, variability in the response of the model.
2. Cell culture on multiwell plates
   1. Seed the Caco-2 cells at a density of 50,000 cells/cm² in 6-well collagen-coated plates.
      NOTE: This step usually works best if done on a Wednesday. The following steps will make it evident why this day of the week is optimal.
   2. Grow the cells for 12 days at 37 °C in an incubator with a 5% CO₂ air atmosphere (constant humidity) using DMEM supplemented with 25 mM HEPES (pH 7.2), 10% (v/v) FBS, and 1% antibiotic-antimycotic solution.
      NOTE: Culturing the cell monolayers longer than 12 days can result in cell overgrowth. Previous research has clearly shown that, under these conditions, at 12 days post-seeding, the cell monolayer is mature, attached well to the plate, and optimal in the consistency of response. Growing the cells longer, such as up to 19-21 days, results in cell overgrowth, and the media rapidly depletes in nutrients, resulting in unhealthy monolayers.
   3. During the 12-day period, change the medium at least every 2 days on a consistent daily schedule.
   4. On the 12^th day post seeding, replace the culture medium with 2 mL of Minimum Essential Medium (MEM [pH 7]) supplemented with 10 mM PIPES (piperazine-N,N'-bis-[2-ethanesulfonic acid]), 1% antibiotic-antimycotic solution, hydrocortisone (4 mg/L), insulin (5 mg/L), selenium (5 μg/L), triiodothyronine (34 μg/L), and epidermal growth factor (20 μg/L).
      NOTE: If seeding was started on a Wednesday, then the 12th day would be a Monday.
   5. On the following day (i.e., day 13), remove the MEM and replace it with 1 mL of MEM (pH 7).
      NOTE: This step would occur on a Tuesday. This is the day when the bioassay begins; thus, the 13-day schedule yields the advantage of a consistent weekly schedule, allowing the bioassay to be conducted consistently on the same weekday.

4. In vitro digestion

1. Preparation of insert rings
   1. Create a sterilized insert ring using a silicone O-ring fitted with an acid-washed dialysis membrane (Figure 1A).
      NOTE: Prepare inserts 1 day in advance (i.e., Monday, day 12) of the day of the bioassay and store in 18 MΩ water at 4 °C until ready to use.
   2. On the day of the bioassay (Tuesday, day 13), remove the inserts from the refrigerator, drain, and replace the water with 0.5 M HCl. Leave it at room temperature in a laminar flow hood for at least 1 h prior to use.
      NOTE: This step should be done prior to removing the MEM from the culture plates. Acid washing of the membrane serves to remove possible contaminating Fe and sterilizes the insert ring and membrane.
   3. Drain the 0.5 M HCl from the inserts and rinse with sterile 18 MΩ water. Store in sterile 18 MΩ water at room temperature in a laminar flow hood until ready to use.
   4. Insert a ring into each well of the 6-well plates with Caco-2 cells, thereby creating a two-chamber system. Return the plates with the inserts to the incubator.
      NOTE: This step should be done just after fresh 1.0 mL of MEM is added to each well (see Step 3.2.5.).
2. Preparation of pepsin solution
   1. On the day of the experiment, prepare the pepsin solution by dissolving 0.145 g of pepsin in 50 mL of 0.1 M HCl. Shake the solution gently on a platform shaker for 30 min at room temperature.
3. Preparation of pancreatin-bile solution
   1. On the same day as the experiment, prepare 0.1 M NaHCO₃ by dissolving 2.1 g of NaHCO₃ in 250 mL of 18 MΩ water.
   2. Mix 0.35 g of pancreatin and 2.1 g of bile extract in 175 mL of 0.1 M NaHCO₃.
   3. Once the pancreatin and bile extract are solubilized, add 87.5 g of a weak cation exchange resin (see the Table of Materials) and mix by shaking for 30 min at room temperature.
   4. Pour the slurry into a large column and collect the eluate.
   5. Elute the column with an additional 70 mL of 0.1 M NaHCO₃, collecting this volume into the pancreatin bile solution.
      NOTE: The purpose of the resin is to remove contaminant Fe commonly found in the pancreatin-bile extracts.
4. Initiate in vitro digestion.
   1. Weigh out the sample in a sterile 50 mL centrifuge tube (polypropylene), followed by the addition of 10 mL of physiological saline at pH 2, containing 140 mM NaCl and 5 mM KCl.
   2. Initiate the gastric digestion process by adding 0.5 mL of the prepared porcine pepsin solution to the sample and incubate on a rocking shaker at a low, gentle setting for 1 h at 37 °C.
   3. Following this period, initiate the intestinal digestion process of each sample by adjusting the pH to 5.5-6.0 with 1.0 M NaHCO₃.
   4. Add 2.5 mL of the pancreatin-bile solution to each sample tube and adjust the pH to 6.9-7.0 with 1.0 M NaHCO₃.
   5. Once the pH is adjusted, equalize the volume in each tube using 140 mM NaCl, 5 mM KCl (pH 6.7) solution, measuring the weight of the tube with a target value of 15 g.
      NOTE: For some foods, one may need to bring the total volume to 16 g or 17 g, depending on the buffering capacity of the foods.
   6. Transfer 1.5 mL of each intestinal digest into the upper chamber (i.e., containing the insert ring with the dialysis membrane) of a corresponding well of the 6-well culture plate containing the Caco-2 cells (Figure 1B).
   7. Replace the plate cover and incubate at 37 °C (5% CO₂ air atmosphere) on a rocking shaker at 6 oscillations/min for 2 h.
   8. Remove the insert ring with the digest and add an additional 1 mL of MEM (pH 7) to each well.
   9. Return the cell culture plate to the incubator (37 °C; 5% CO₂ air atmosphere) for an additional 22 h.
   10. After 22 h, remove the cell culture medium and add 2.0 mL of 18 MΩ water to the cell monolayer.
       NOTE: The water will osmotically lyse the cells.
   11. Harvest the entire cell lysate into standard polypropylene microcentrifuge tubes or similar for subsequent cell protein and cell ferritin analyses.