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

Published: April 30, 2023

Abstract

Source: Glahn, R. P. The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability. J. Vis. Exp. (2022)

This video demonstrates an in vitro technique to assess iron bioavailability in complex food sources. The technique combines the simulation of human digestion along with iron uptake by differentiated Caco-2 cells. Intracellular ferritin formation acts as a marker for cellular iron uptake.

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…

Representative Results

Figure 1: Insert ring setup for Caco-2 cell Fe uptake. (A) Image of Caco-2 cells and insert ring with attached dialysis membrane. (B) Diagram of the overall procedure for in vitro digestion coupled with Caco-2 cell Fe uptake within a single well of the multi-well plate.

Divulgaciones

The authors have nothing to disclose.

Materials

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
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
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

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Caco-2 Cell Bioassay: An In Vitro Method for Measuring Iron Bioavailability in Complex Food Sources. J. Vis. Exp. (Pending Publication), e21211, doi: (2023).

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