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The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability
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Optimizing INFOGEST Digest Conditioning for Reliable In Vitro Assessment of Nutrient Bioavailability Using Caco-2

Giulia Camporesi1, Carlo Canzian2, Alessandra Bordoni2

  • 1Interdepartmental Centre for Industrial Agri-Food Research (CIRI), University of Bologna, Piazza Goidanich 60, 47521 Cesena, Italy.

Nutrients
|January 28, 2026
PubMed
Summary
This summary is machine-generated.

Ultrafiltration using 3 kDa membranes effectively preserves intestinal cell viability and barrier function in digested foods. This method enhances the reliability of in vitro bioavailability studies by improving digest compatibility with intestinal models.

Keywords:
Caco-2 cellsINFOGESTcell viabilitydigest conditioningdigestionepithelial barrier integrityin vitro bioavailability

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Area of Science:

  • Nutrition Science
  • In Vitro Digestion Models
  • Cell Biology

Background:

  • In vitro assessment of nutrient bioavailability often uses gastrointestinal digestion models coupled with intestinal cell systems.
  • Static digestion protocols like INFOGEST can compromise epithelial barrier integrity, limiting study reproducibility.
  • Standardized methods are needed to prepare digests for reliable in vitro testing.

Purpose of the Study:

  • To compare five digest conditioning strategies for preserving intestinal epithelial cell viability and barrier function.
  • To identify the optimal method for preparing INFOGEST digests for use with intestinal cell models.
  • To ensure physiologically relevant concentrations and reproducible exposure conditions.

Main Methods:

  • Systematic comparison of acidification, centrifugation, rapid freezing, and ultrafiltration (10 kDa and 3 kDa MWCO).
  • Evaluation of INFOGEST digests from yogurt, biscuit, and canned mackerel.
  • Assessment of Caco-2 cell viability (MTT assay) and barrier integrity (TEER measurements).

Main Results:

  • 3 kDa ultrafiltration consistently preserved epithelial viability and barrier integrity across all food matrices at a 1:10 dilution.
  • Other conditioning methods failed to maintain transepithelial electrical resistance (TEER) despite acceptable cell viability.
  • Food matrix effects influenced results at lower dilutions, necessitating matrix-specific evaluation.

Conclusions:

  • 3 kDa ultrafiltration is an effective, minimally invasive strategy for improving INFOGEST digest compatibility with intestinal cell models.
  • This method enables reproducible exposure conditions that maintain epithelial integrity.
  • The findings support more reliable in vitro bioavailability assessments and methodological standardization in nutrition research.