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

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 ....

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 sampl.......

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.......

Materials

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