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