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).