BioUptake and BioAbsorption via Caco-2 Permeability Assay

In vitro gold standard assay for predicting bioabsorption and biouptake by human intestines.

Why | Significance

Many substances are absorbed by passive transport through the intestine membrane into blood. Such transcellular permeability properties can significantly influence the oral absorption of a food/nutritional product or drug candidate. Intestinal permeability is a critical characteristic that determines the rate and extent of human absorption and ultimately affects the bioavailability of a nutritional product or drug candidate.

What are Caco-2 cells?

Caco-2 cells are human intestinal cells that are widely used to measure biouptake and absorption capability of a food and nutritional product. When cultured under specific conditions they become differentiated and polarized such that morphologically and functionally resemble the epithelial cells of human small intestine, and the monolayers of caco-2 cells resemble the membrane of human intestines.

What is Caco-2 Permeability Assay? – Predict Human Intestinal Bioabsorption and Biouptake of a Substance

The Caco-2 permeability assay is considered to be the golden standard method in nutraceutical and pharmaceutical industries for in vitro prediction of in vivo human intestinal bioabsorption and biouptake of a food/nutritional product or medicine candidate. In drug discovery field, Caco-2 permeability assay is also used to predict drug-drug interactions (DDIs) of orally administered drugs. The FDA recommends that drug-drug interactions should be performed during drug development (FDA Guidance for Pharmaceutical Industry).

More Facts About Caco-2 Permeability Assay

• The Caco-2 Permeability Assay utilizes a Caco-2 cell layer grown on a supportive membrane surface that separates two compartments. Typically, the substance to be studied is added to the apical side of a confluent monolayer of Caco-2 cells and permeability is measured by monitoring the appearance of the test compound on the opposite side (basolateral side) of the monolayer.
• Bi-directional Caco-2 permeability assay can also be performed if requested. If performing a bi-directional assay, the efflux ratio (ER) will also be calculated from the ratio of B-to-A and A-to-B permeability.
• If requested, the P-glycoprotein (P-gp) inhibitor can be included to identify whether active transport is mediated by P-gp

Summary of Assay Parameters:

• Caco-2 passage number: 40-60
• Period of cell culture: 21 days
• Incubation time: 2 hours
• Temperature: 37 degree Celsius
• Number of replicates: 2 or as requested
• Integrity Marker: Lucifer Yellow
• Control Compounds: Propranolol, atenolol (Additional controls/comparison substances can be requested)
• Analysis Method: high-performance liquid chromatography/mass spectroscopy (HPLC-MS/MS) or as custom-designed

Requesting a Caco-2 Permeability Assay

• Amount of Compound Requested: substances in solid or solvent phase are both acceptable
• Pure compound: 5mg (small molecules) or 50mg (large molecules/peptides)
• Mixture substance: 20ml (liquid) or 20g (solid)
• Information of the Test Substance Requested:
• Pure compound: molecular structure or molecular weight
• Mixture substance: marker molecule(s) to be monitored. Identification of marker molecule(s) is provided if requested.
• Turnaround Time: 4-6 weeks (2 weeks for routine testing)