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Summary

The Caco-2 cell bioassay for iron (Fe) bioavailability represents a cost-effective and versatile approach to assess Fe bioavailability from foods, food products, supplements, meals, and even diet regimens. Thoroughly validated to human studies, it represents the state of the art for studies of Fe bioavailability.

Abstract

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte.

Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

Introduction

To fully understand the research need and benefit of the Caco-2 cell bioassay for Fe bioavailability, one must first understand the approaches that were in place prior to the advent of this model. The measurement of Fe bioavailability from a food or meal in vivo is a challenging task, particularly when combinations of food need to be assessed in a meal or diet. Isotopic labeling has been the most common approach for the measurement of Fe bioavailability over the past 50 years1. Isotopic labeling is used for single-meal and multiple-meal studies and is impractical for long-term studies. Stable isotopes of Fe such as 57Fe and 58Fe are the most commonly used; however, studies have been conducted with radioisotopes such as 59Fe, utilizing whole-body counting2. For plant foods, isotopic labeling has been done via extrinsic or intrinsic labeling. For extrinsic labeling, a known amount of isotope is added to the food or meal. The food is then mixed, and a 15-30 min equilibration period is incorporated into the protocol prior to the consumption. Hydroponic culture-adding the isotope to the nutrient solution to incorporate it into the plant while it grows and develops-is required for the intrinsic labeling of plant foods. The pros and cons of each approach are discussed below.

Extrinsic isotopic labeling
In the early to mid-1970s, human Fe absorption was studied by extrinsic labeling of Fe in foods, wherein a known amount of isotope is added to the known amount of Fe in the food or meal, mixed, and equilibrated for 15-30 min before measurements. Various amounts of extrinsic isotopes have been used, ranging from 1% to 100% of the intrinsic Fe, but most commonly in the range of 7%-30%3. Extrinsic labeling is based on the assumption that the extrinsic Fe isotope gets fully equilibrated with the intrinsic Fe of the food or meal. Extrinsic isotope absorption is then measured, and each atom of the extrinsic isotope is calculated to represent a given number of intrinsic Fe atoms. This calculation is based on the relative molar amounts. In 1983, multiple validation studies of the technique were summarized in a review paper4. Validation of the technique was done by simultaneously comparing the percent absorption of the extrinsic isotopic label to the percent absorption of an intrinsic isotopic label. Thus, a ratio of the extrinsic to intrinsic absorption close to 1 suggests that each pool of Fe was equally absorbed. At the time, a ratio close to 1 was also considered to represent equilibration of the extrinsic isotope with the intrinsic Fe of the food or meal. Ratios of extrinsic to intrinsic Fe absorption ranged from mean values of 0.40 to 1.62, with a mean (±SD) ratio of 1.08 ± 0.14 in 63 comparisons. It is important to note that, in all of the studies summarized in this review, none directly tested the equilibration of the extrinsic label with the intrinsic Fe. In summary, the authors of the review concluded the following:

"The extrinsic tag technique has proven valid for several foods under certain experimental conditions. But, this method cannot yet be considered proven with regards to all types of foods. The extrinsic tag method is not appropriate for monitoring iron absorption from a diet that contains insoluble forms of iron. The validity of this technique relies upon the basic assumption that the extrinsic tag exchanges completely with all endogenous nonheme food iron. At present it is not known how completely the different forms of nonheme iron are labeled by an extrinsic tag. This is important in light of studies which have suggested that iron inhibitors may affect the extrinsic tag differently than some forms of nonheme iron in foods. Research on food factors which can impair a complete isotopic exchange is scant. Thus, interpretation of bioavailability data from extrinsic tag research requires consideration of inhibitors of exchange which may be present in the food or diet."

Since 1983, only two studies have been published that evaluated the accuracy of extrinsic labeling of Fe3,5. In both these studies, the equilibration of an extrinsic isotopic label was directly compared with the intrinsic Fe of the foods, which, in these studies, were staple food crops. White, red, and black bean varieties were tested, along with lentils and maize. Using established in vitro digestion techniques and the measurement of Fe solubility and precipitation, both studies demonstrated that extrinsic isotopic labeling does not consistently result in full equilibration, with evidence that, for some bean varieties, the misequilibration can be very high depending on the amount of extrinsic isotope and seed coat color3. Despite the conclusions of the 1983 review paper, extrinsic labeling studies of beans continued6,7,8,9,10,11,12. None of these studies included testing the equilibration of the extrinsic label with the intrinsic Fe.

Intrinsic labeling
Intrinsic labeling of plant food for the assessment of Fe bioavailability eliminates the accuracy issues of equilibration in extrinsic labeling. However, this approach cannot yield large amounts of material because of the requirement of greenhouse space for hydroponic culture. Hydroponic culture is labor-intensive, requires a high quantity of expensive stable isotope, and often results in plant growth different in terms of yield and seed Fe concentration. Due to the cost, intrinsic labeling is only suitable for small-scale studies aimed at understanding mechanisms underlying Fe uptake or factors influencing Fe uptake from foods. Production of 1-2 kg of a staple food crop costs approximately $20,000-$30,000 for materials alone, depending on the isotope and hydroponic approach13,14.

Given the challenges associated with isotopic labeling, investigators sought to develop in vitro approaches. Early methods utilized simulated gastric and intestinal food, coupled with the measurement of Fe solubility or Fe dialyzability as an estimate of bioavailability15. Such studies quickly found that Fe dialyzability was not a consistent measure of bioavailability as Fe can be soluble, tightly bound to compounds and, therefore, not exchangeable, leading to the overestimation of bioavailability. To address these issues, methodology to utilize a human intestinal cell line evolved, thereby adding a living component and enabling the measurement of Fe uptake16. The human intestinal cells-Caco-2 cells-originated from a human colon carcinoma and have been widely used in nutrient uptake studies. This cell line is useful as, in culture, the cells differentiate into enterocytes that function similarly to the brush border cells of the small intestine. Studies have shown that Caco-2 cells exhibit the appropriate transporters and response to factors that influence Fe uptake17,18.

The initial studies, utilizing radioisotopes to measure Fe uptake in Caco-2 cells, were refined to measure Fe uptake based on Caco-2 cell ferritin formation. Caco-2 cell ferritin measurement enhanced sample throughput and negated issues of radioisotope handling and the equilibration of extrinsic Fe with intrinsic Fe19,20. Measurement of Fe uptake via ferritin formation enabled researchers to study a broad range of foods, including complex meals21. Thus, simulated (in vitro) digestion coupled with Caco-2 cell Fe uptake provided a better physiological assessment of Fe uptake from foods. It is important to note that this model primarily determines relative differences in Fe bioavailability. Like many useful cell lines, Caco-2 cells also have shown variability in responsiveness but have maintained consistent relative differences in Fe uptake between foods. Proper technique and careful attention to detail can improve consistent cell ferritin formation response in Caco-2 cells.

The in vitro digestion/Caco-2 cell model is also known as Caco-2 cell bioassay. This assay has been thoroughly validated via direct comparison to human and animal studies22. In addition to the direct parallel comparison of the bioassay to human efficacy trials, this model has been shown to exhibit a qualitatively similar response in Fe uptake to that of humans18,19,23. Therefore, as an in vitro approach, the Caco-2 cell bioassay warrants high credibility as a screening tool for evaluating Fe nutrition from foods. It has been widely applied to numerous foods and food products21,24,25,26,27,28.

Since its inception in 1998, the Caco-2 cell bioassay has advanced the field of Fe nutrition as it has helped identify factors that influence intestinal Fe uptake. In so doing, this model has developed and refined research objectives for more definitive and less costly human studies. One could also argue that the use of the model negates the need for some human trials.

In summary, the relative delivery of Fe from a food or meal can be measured with the Caco-2 cell bioassay. Regardless of the amount of Fe in the test meal, the bioassay defines the relative amount of Fe taken up into the enterocyte-the first step of the absorption process. This is the most important step in defining Fe bioavailability, as most often the goal is to measure with the intent to improve or, at the very least, monitor the nutritional quality of Fe in a food. Given that iron status is regulated by absorption, and thus Fe uptake is upregulated in Fe-deficient individuals to meet nutritional needs, the standard conditions of the model are designed so that Fe uptake by the cells is maximal. In this way, the bioassay provides a true measure of the potential of the food to deliver Fe.

Protocol

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 response29,30,31. 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 12th 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.

5. Measurement of Caco-2 cell ferritin and cell protein

  1. Use the cell lysate from Step 4.4.10. for the measurement of cell ferritin and protein.
    1. To measure the ferritin content of the Caco-2 cells, follow the kit's instructions (see the Table of Materials), with the exception of increasing the incubation time from 30 min to 2 h for the mouse anti-ferritin antibody-horseradish peroxidase (HRP) conjugate.
    2. To measure cell protein, follow the instructions provided in the cell protein kit (see the Table of Materials).

Results

Identification and measurement of Fe bioavailability in staple food crops
One of the primary reasons for developing this model was to identify factors that influence Fe bioavailability in staple food crops and provide a tool for plant breeders that would enable them to identify and develop varieties with enhanced Fe bioavailability. The common bean (Phaseolus vulgaris) has been targeted globally as a crop for Fe biofortification; thus, the model has been applied extensively to evaluate the...

Discussion

Since its inception, numerous studies have been published that describe this method for the Caco-2 cell bioassay. The basic conditions have remained relatively unchanged since the initial publication in 199818. However, over the past 20 years, numerous technical details have been refined and standardized to yield unprecedented consistency in the response of the bioassay. Careful and precise adherence to the cell culture and in vitro digestion conditions are the key to the consistent and s...

Disclosures

The author has no conflicts of interest.

Acknowledgements

The author is deeply grateful for the technical efforts of Yongpei Chang and Mary Bodis. The extremely successful application of this model in the field of nutrition is a direct result of their expertise and attention to detail. The development of this model was funded entirely by the United States Department of Agriculture, Agricultural Research Service.

Materials

NameCompanyCatalog NumberComments
0.5 M HClFisher ScientificA508-4 Hydrochloric Acid TraceMetal Grade
18 megaohm waterAlso known as distilled, deionized water
3,3′,5-Triiodo-L-thyronine sodium saltSigma Aldrich CoT6397
6-well platesCostar3506Use for bioassay experiments
ascorbic acidSigma Aldrich CoA0278
bile extractSigma Aldrich CoB8631
Caco-2 cellsAmerican Type Culture CollectionHTB-37HTB-37 is a common variety.
Cell culture flasks T225Falcon 353138
Cell culture flasks T25Corning430639
Cell culture flasks T75Corning430641U
Chelex-100Bio-Rad Laboratories Inc142832Known as the weak cation exchange resin in the protocol
collagenCorning354236
dialysis membraneSpectrum LaboratoriesSpectra/Por 7 Pretreated RC Dialysis Tubing 15,000 MWCOSpectra/Por 7 Pretreated RC Dialysis Tubing 15,000 MWCO
Dulbecco’s Modified Eagle’s MediumGibco12100046DMEM
epidermal growth factorSigma Aldrich CoE4127-5X.1MG
Ferritin ELISA Assay KitEagle BiosciencesFRR31-K01
fetal bovine serumR&D SystemsS12450Optima
HEPESSigma Aldrich CoH3375
Hydrocortisone-Water SolubleSigma Aldrich CoH0396
insert ringCorning Costarnot soldTranswell, for 6 well plate, without membrane
insulinSigma Aldrich CoI2643
KClSigma Aldrich CoP9333
large columnVWR InternationalKT420400-1530
Minimum Essential MediumGibco41500034MEM
NaClFisher ScientificS271
pancreatinSigma Aldrich CoP1750
PIPES disodium saltSigma Aldrich CoPiperazine-1,4-bis(2-ethanesulfonic acid) disodium salt P3768
porcine pepsinSigma Aldrich CoP6887 or (P7012-25G Sigma
protein assay kitBio-Rad Laboratories IncBio-Rad DC protein assay kit 500-0116Measurement of Caco-2 cell protein
silicone o ringsWeb Seal, Inc Rochester NY2-215S500
sodium bicarbonateFisher ScientificS233
Sodium seleniteSigma Aldrich CoS5261
ZellShieldMinerva Biolabs13-0050Use at 1% as antibiotic/antimycotic ordered through Thomas Scientific

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Caco 2 Cell BioassayIron BioavailabilityNutritional QualityHigh Throughput ApproachCell CultureDMEM MediumAntibiotic Antimycotic SolutionCollagen Coated PlatesIncubation ConditionsMinimum Essential MediumSilicone O ringDialysis MembranePancreatin bile SolutionCation Exchange ResinCentrifugation

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