Name: the caco-2 cell bioassay for measurement of food iron bioavailability
Uploaded: 2022-04-28T14:15:00
Description: Watch this Scientific Journal Video about The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability at JoVE.com

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.

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 sampl...

<ol>
	<li>Fairweather-Tait, S. J., Dainty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+stable+isotopes+to+assess+the+bioavailability+of+trace+elements:+a+review.">Use of stable isotopes to assess the bioavailability of trace elements: a review.</a> Food Additives Contaminants. 19 (10), 939-947 (2002).</li><li>Hadley, K. B., Johnson, L. K., Hunt, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+absorption+by+healthy+women+is+not+associated+with+either+serum+or+urinary+prohepcidin.">Iron absorption by healthy women is not associated with either serum or urinary prohepcidin.</a> American Journal of Clinical Nutrition. 84 (1), 150-155 (2006).</li><li>Glahn, R. P., Cheng, Z., Giri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+labeling+of+staple+food+crops+with+isotopic+iron+does+not+consistently+result+in+full+equilibration:+revisiting+the+methodology.">Extrinsic labeling of staple food crops with isotopic iron does not consistently result in full equilibration: revisiting the methodology.</a> Journal of Agricultural and Food Chemistry. 63 (43), 9621-9628 (2015).</li><li>Consaul, J. R., Lee, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+tagging+in+iron+bioavailability+research:+a+critical+review.">Extrinsic tagging in iron bioavailability research: a critical review.</a> Journal of Agricultural and Food Chemistry. 31 (4), 684-689 (1983).</li><li>Jin, F., Cheng, Z., Rutzke, M. A., Welch, R. M., Glahn, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+labeling+method+may+not+accurately+measure+Fe+absorption+from+cooked+pinto+beans+(Phaseolus+vulgaris):+comparison+of+extrinsic+and+intrinsic+labeling+of+beans.">Extrinsic labeling method may not accurately measure Fe absorption from cooked pinto beans (Phaseolus vulgaris): comparison of extrinsic and intrinsic labeling of beans.</a> Journal of Agricultural and Food Chemistry. 56 (16), 6881-6885 (2008).</li><li>Junqueira-Franco, M. V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+absorption+from+beans+with+different+contents+of+iron,+evaluated+by+stable+isotopes.">Iron absorption from beans with different contents of iron, evaluated by stable isotopes.</a> Clinical Nutrition ESPEN. 25, 121-125 (2018).</li><li>Petry, N., Egli, I., Zeder, C., Walczyk, T., Hurrell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphenols+and+phytic+acid+contribute+to+the+low+iron+bioavailability+from+common+beans+in+young+women.">Polyphenols and phytic acid contribute to the low iron bioavailability from common beans in young women.</a> Journal of Nutrition. 140 (11), 1977-1982 (2010).</li><li>Petry, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+iron+isotope+studies+in+Rwandese+women+indicate+that+the+common+bean+has+limited+potential+as+a+vehicle+for+iron+biofortification.">Stable iron isotope studies in Rwandese women indicate that the common bean has limited potential as a vehicle for iron biofortification.</a> Journal of Nutrition. 142 (3), 492-497 (2012).</li><li>Petry, N., Egli, I., Campion, B., Nielsen, E., Hurrell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+reduction+of+phytate+in+common+bean+(Phaseolus+vulgaris+L.)+seeds+increases+iron+absorption+in+young+women.">Genetic reduction of phytate in common bean (Phaseolus vulgaris L.) seeds increases iron absorption in young women.</a> Journal of Nutrition. 143 (8), 1219-1224 (2013).</li><li>Petry, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytic+acid+concentration+influences+iron+bioavailability+from+biofortified+beans+in+Rwandese+women+with+low+iron+status.">Phytic acid concentration influences iron bioavailability from biofortified beans in Rwandese women with low iron status.</a> Journal of Nutrition. 144 (11), 1681-1687 (2014).</li><li>Petry, N., Boy, E., Wirth, J. P., Hurrell, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+The+potential+of+the+common+bean+(Phaseolus+vulgaris)+as+a+vehicle+for+iron+biofortification.">Review: The potential of the common bean (Phaseolus vulgaris) as a vehicle for iron biofortification.</a> Nutrients. 7 (2), 1144-1173 (2015).</li><li>Petry, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Rwandese+women+with+low+iron+status,+iron+absorption+from+low-phytic+acid+beans+and+biofortified+beans+is+comparable,+but+low-phytic+acid+beans+cause+adverse+gastrointestinal+symptoms.">In Rwandese women with low iron status, iron absorption from low-phytic acid beans and biofortified beans is comparable, but low-phytic acid beans cause adverse gastrointestinal symptoms.</a> Journal of Nutrition. 146 (5), 970-975 (2016).</li><li>Donangelo, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+and+zinc+absorption+from+two+bean+(Phaseolus+vulgaris+L.)+genotypes+in+young+women.">Iron and zinc absorption from two bean (Phaseolus vulgaris L.) genotypes in young women.</a> Journal of Agricultural and Food Chemistry. 51 (17), 5137-5143 (2003).</li><li>Dellavalle, D. M., Glahn, R. P., Shaff, J. E., O'Brien, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+absorption+from+an+intrinsically+labeled+lentil+meal+is+low+but+upregulated+in+women+with+poor+iron+status.">Iron absorption from an intrinsically labeled lentil meal is low but upregulated in women with poor iron status.</a> Journal of Nutrition. 145 (10), 2253-2257 (2015).</li><li>Miller, D. D., Schricker, B. R., Rasmussen, R. R., Van Campen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+method+for+estimation+of+iron+availability+from+meals.">An in vitro method for estimation of iron availability from meals.</a> American Journal of Clinical Nutrition. 34 (10), 2248-2256 (1981).</li><li>Glahn, R. P., Wien, E. M., Van Campen, D. R., Miller, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caco-2+cell+iron+uptake+from+meat+and+casein+digests+parallels+in+vivo+studies:+use+of+a+novel+in+vitro+method+for+rapid+estimation+of+iron+bioavailability.">Caco-2 cell iron uptake from meat and casein digests parallels in vivo studies: use of a novel in vitro method for rapid estimation of iron bioavailability.</a> Journal of Nutrition. 126 (1), 332-339 (1996).</li><li>Martini, L. A., Tchack, L., Wood, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+treatment+downregulates+DMT1+and+IREG1+mRNA+expression+in+Caco-2+cells.">Iron treatment downregulates DMT1 and IREG1 mRNA expression in Caco-2 cells.</a> Journal of Nutrition. 132 (4), 693-696 (2002).</li><li>Glahn, R. P., Wortley, G. M., South, P. K., Miller, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+iron+uptake+by+phytic+acid,+tannic+acid,+and+ZnCl2:+studies+using+an+in+vitro+digestion/Caco-2+cell+model.">Inhibition of iron uptake by phytic acid, tannic acid, and ZnCl2: studies using an in vitro digestion/Caco-2 cell model.</a> Journal of Agricultural and Food Chemistry. 50 (2), 390-395 (2002).</li><li>Glahn, R. P., Lee, O. A., Yeung, A., Goldman, M. I., Miller, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caco-2+cell+ferritin+formation+predicts+nonradiolabeled+food+iron+availability+in+an+in+vitro+digestion/Caco-2+cell+culture+model.">Caco-2 cell ferritin formation predicts nonradiolabeled food iron availability in an in vitro digestion/Caco-2 cell culture model.</a> Journal of Nutrition. 128 (9), 1555-1561 (1998).</li><li>Yun, S., Habicht, J. P., Miller, D. D., Glahn, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+digestion/Caco-2+cell+culture+system+accurately+predicts+the+effects+of+ascorbic+acid+and+polyphenolic+compounds+on+iron+bioavailability+in+humans.">An in vitro digestion/Caco-2 cell culture system accurately predicts the effects of ascorbic acid and polyphenolic compounds on iron bioavailability in humans.</a> Journal of Nutrition. 134 (10), 2717-2721 (2004).</li><li>Pach&#243;n, H., Stoltzfus, R. J., Glahn, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogenization,+lyophilization+or+acid-extraction+of+meat+products+improves+iron+uptake+from+cereal-meat+product+combinations+in+an+in+vitro+digestion/Caco-2+cell+model.">Homogenization, lyophilization or acid-extraction of meat products improves iron uptake from cereal-meat product combinations in an in vitro digestion/Caco-2 cell model.</a> British Journal of Nutrition. 101 (6), 816-821 (2009).</li><li>Tako, E., Bar, H., Glahn, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combined+application+of+the+Caco-2+cell+bioassay+coupled+with+in+vivo+(Gallus+gallus)+feeding+trial+represents+an+effective+approach+to+predicting+Fe+bioavailability+in+humans.">The combined application of the Caco-2 cell bioassay coupled with in vivo (Gallus gallus) feeding trial represents an effective approach to predicting Fe bioavailability in humans.</a> Nutrients. 8 (11), 732-757 (2016).</li><li>Engle-Stone, R., Yeung, A., Welch, R., Glahn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meat+and+ascorbic+acid+can+promote+Fe+availability+from+Fe-phytate+but+not+from+Fe-tannic+acid+complexes.">Meat and ascorbic acid can promote Fe availability from Fe-phytate but not from Fe-tannic acid complexes.</a> Journal of Agricultural and Food Chemistry. 53 (26), 10276-10284 (2005).</li><li>Tako, E., Blair, M. W., Glahn, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofortified+red+mottled+beans+(Phaseolus+vulgaris+L.)+in+a+maize+and+bean+diet+provide+more+bioavailable+iron+than+standard+red+mottled+beans:+studies+in+poultry+(Gallus+gallus)+and+an+in+vitro+digestion/Caco-2+model.">Biofortified red mottled beans (Phaseolus vulgaris L.) in a maize and bean diet provide more bioavailable iron than standard red mottled beans: studies in poultry (Gallus gallus) and an in vitro digestion/Caco-2 model.</a> Nutrition Journal. 10, 113 (2011).</li><li>Pach&#243;n, H., Stoltzfus, R. J., Glahn, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+thigh,+chicken+liver,+and+iron-fortified+wheat+flour+increase+iron+uptake+in+an+in+vitro+digestion/Caco-2+cell+model.">Chicken thigh, chicken liver, and iron-fortified wheat flour increase iron uptake in an in vitro digestion/Caco-2 cell model.</a> Nutrition Research. 28 (12), 851-858 (2008).</li><li>Beasley, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+engineering+of+bread+wheat+improves+grain+iron+concentration+and+bioavailability.">Metabolic engineering of bread wheat improves grain iron concentration and bioavailability.</a> Plant Biotechnology Journal. 17 (8), 1514-1526 (2019).</li><li>Zhu, L., Glahn, R. P., Nelson, D., Miller, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+soluble+ferric+pyrophosphate+to+common+iron+salts+and+chelates+as+sources+of+bioavailable+iron+in+a+Caco-2+cell+culture+model.">Comparing soluble ferric pyrophosphate to common iron salts and chelates as sources of bioavailable iron in a Caco-2 cell culture model.</a> Journal of Agricultural and Food Chemistry. 57 (11), 5014-5019 (2009).</li><li>Wortley, G., Leusner, S., Good, C., Gugger, E., Glahn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+availability+of+a+fortified+processed+wheat+cereal:+a+comparison+of+fourteen+iron+forms+using+an+in+vitro+digestion/human+colonic+adenocarcinoma+(Caco-2)+cell+model.">Iron availability of a fortified processed wheat cereal: a comparison of fourteen iron forms using an in vitro digestion/human colonic adenocarcinoma (Caco-2) cell model.</a> British Journal of Nutrition. 93 (1), 65-71 (2005).</li><li>Jumarie, C., Malo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alkaline+phosphatase+and+peptidase+activities+in+Caco-2+cells:+differential+response+to+triiodothyronine.">Alkaline phosphatase and peptidase activities in Caco-2 cells: differential response to triiodothyronine.</a> In Vitro Cellular and Developmental Biology - Animal. 30 (11), 753-760 (1994).</li><li>Engle, M. J., Goetz, G. S., Alpers, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caco-2+cells+express+a+combination+of+colonocyte+and+enterocyte+phenotypes.">Caco-2 cells express a combination of colonocyte and enterocyte phenotypes.</a> Journal of Cellular Physiology. 174 (3), 362-369 (1998).</li><li>Ferruzza, S., Rossi, C., Sambuy, Y., Scarino, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-reduced+and+serum-free+media+for+differentiation+of+Caco-2+cells.">Serum-reduced and serum-free media for differentiation of Caco-2 cells.</a> Alternatives to Animal Experimentation. 30 (2), 159-168 (2013).</li><li>Wiesinger, J. A., Cichy, K. A., Tako, E., Glahn, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fast+cooking+and+enhanced+iron+bioavailability+properties+of+the+Manteca+yellow+bean+(Phaseolus+vulgaris+L).">The fast cooking and enhanced iron bioavailability properties of the Manteca yellow bean (Phaseolus vulgaris L).</a> Nutrients. 10 (11), 1609 (2018).</li><li>Wiesinger, J. A., Cichy, K. A., Hooper, S. D., Hart, J. J., Glahn, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+white+or+yellow+dry+beans+(Phaseolus+vulgaris+L.)+into+a+heat+treated+flour+enhances+the+iron+bioavailability+of+bean-based+pastas.">Processing white or yellow dry beans (Phaseolus vulgaris L.) into a heat treated flour enhances the iron bioavailability of bean-based pastas.</a> Journal of Functional Foods. 71, 104018 (2020).</li></ol>

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...

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 (&#177;SD) ratio of 1.08 &#177; 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:
&#34;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.&#34;
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.

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		<tr>
			<td>0.5 M HCl</td>
			<td>Fisher Scientific</td>
			<td>A508-4 Hydrochloric Acid TraceMetal Grade</td>
			<td></td>
		</tr>
		<tr>
			<td>18 megaohm water</td>
			<td></td>
			<td></td>
			<td>Also known as distilled, deionized water</td>
		</tr>
		<tr>
			<td>3,3&#8242;,5-Triiodo-L-thyronine sodium salt</td>
			<td>Sigma Aldrich Co</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Costar</td>
			<td>3506</td>
			<td>Use for bioassay experiments</td>
		</tr>
		<tr>
			<td>ascorbic acid</td>
			<td>Sigma Aldrich Co</td>
			<td>A0278</td>
			<td></td>
		</tr>
		<tr>
			<td>bile extract</td>
			<td>Sigma Aldrich Co</td>
			<td>B8631</td>
			<td></td>
		</tr>
		<tr>
			<td>Caco-2 cells</td>
			<td>American Type Culture Collection</td>
			<td>HTB-37</td>
			<td>HTB-37 is a common variety.</td>
		</tr>
		<tr>
			<td>Cell culture flasks T225</td>
			<td>Falcon</td>
			<td>&#160;353138</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks T25</td>
			<td>Corning</td>
			<td>430639</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks T75</td>
			<td>Corning</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>Chelex-100</td>
			<td>Bio-Rad Laboratories Inc</td>
			<td>142832</td>
			<td>Known as the weak cation exchange resin in the protocol</td>
		</tr>
		<tr>
			<td>collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis membrane</td>
			<td>Spectrum Laboratories</td>
			<td>Spectra/Por 7 Pretreated RC Dialysis Tubing 15,000 MWCO</td>
			<td>Spectra/Por 7 Pretreated RC Dialysis Tubing 15,000 MWCO</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>Gibco</td>
			<td>12100046</td>
			<td>DMEM</td>
		</tr>
		<tr>
			<td>epidermal growth factor</td>
			<td>Sigma Aldrich Co</td>
			<td>E4127-5X.1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferritin ELISA Assay Kit</td>
			<td>Eagle Biosciences</td>
			<td>FRR31-K01</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>R&#38;D Systems</td>
			<td>S12450</td>
			<td>Optima</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich Co</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone-Water Soluble</td>
			<td>Sigma Aldrich Co</td>
			<td>H0396</td>
			<td></td>
		</tr>
		<tr>
			<td>insert ring</td>
			<td>Corning Costar</td>
			<td>not sold</td>
			<td>Transwell, for 6 well plate, without membrane</td>
		</tr>
		<tr>
			<td>insulin</td>
			<td>Sigma Aldrich Co</td>
			<td>I2643</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma Aldrich Co</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>large column</td>
			<td>VWR International</td>
			<td>KT420400-1530</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium</td>
			<td>Gibco</td>
			<td>41500034</td>
			<td>MEM</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271</td>
			<td></td>
		</tr>
		<tr>
			<td>pancreatin</td>
			<td>Sigma Aldrich Co</td>
			<td>P1750</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES disodium salt</td>
			<td>Sigma Aldrich Co</td>
			<td>Piperazine-1,4-bis(2-ethanesulfonic acid) disodium salt P3768</td>
			<td></td>
		</tr>
		<tr>
			<td>porcine pepsin</td>
			<td>Sigma Aldrich Co</td>
			<td>P6887 or (P7012-25G Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>protein assay kit</td>
			<td>Bio-Rad Laboratories Inc</td>
			<td>Bio-Rad DC protein assay kit 500-0116</td>
			<td>Measurement of Caco-2 cell protein</td>
		</tr>
		<tr>
			<td>silicone o rings</td>
			<td>Web Seal, Inc Rochester NY</td>
			<td>2-215S500</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>Fisher Scientific</td>
			<td>S233</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma Aldrich Co</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>ZellShield</td>
			<td>Minerva Biolabs</td>
			<td>13-0050</td>
			<td>Use at 1% as antibiotic/antimycotic ordered through Thomas Scientific</td>
		</tr>
	</tbody>
</table>

the caco-2 cell bioassay for measurement of food iron bioavailability

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.

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...

Watch this Scientific Journal Video about The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability at JoVE.com

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

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.

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is ...

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.

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