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There is no doubt that oral delivery is the most ideal route of drug administration. Following oral administration, the drug can be absorbed into the systemic circulation through passive and active transport routes across the intestinal wall. The key focus of many drug discovery teams is to develop drugs that can be efficiently absorbed across the intestinal epithelium. Caco-2 cell line is the most common and extensively characterized cell model used to evaluate the absorption of drugs via the intestinal epithelial cells. Caco-2 cells possess many characteristics of primary intestinal epithelial cells in function and morphology. Although Caco-2 cells were originally derived from human colon adenocarcinoma, it has been shown to express various efflux transporters, microvillar transporters, hydrolases, and Phase II conjugation enzymes, which are more representative of enterocytes of the small intestine. Caco-2 cells also express enzymes such as membrane peptidases and disaccharidases. Importantly, Caco-2 cells are able to fully polarize into a differentiated monolayer, displaying brush borders and tight junctions after 21 days of culturing.

Table 1. Characteristics of Caco-2 cell line.

Parameter  Description
OriginHuman colorectal adenocarcinoma
Growth in cultureMonolayer epithelial cells
Differentiation14-21 days after confluence in standard culture medium
MorphologyPolarized cells, with tight junctions, apical brush border
Electrical resistanceHigh transepithelial electrical resistance (TEER)
Digestive enzymesTypical membranous peptidases and disaccharidases of the small intestine
Active transportAmino acids, sugars, vitamins, hormones

The Advantages of Caco-2 Permeability Assay

Caco-2 model has a number of key advantages, which make it the gold standard for drug permeability assays in both academia and more commonly, industry. There are three main advantages to their wide-scale use:

  • Capability for high-throughput manufacture
  • Good reproducibility in vivo
  • Low model variation (high data reliability)

Assay Protocol

Compound Requirements 10 mM DMSO stock
Test Article Concentration 10 µM
Replicates Triplicate
Incubation Time 120 min at 37°C in a CO2 incubator
Analysis Method LC-MS/MS
Control Compounds Digoxin, atenolol, propranolol or other positive controls on request
Cell Monolayer Integrity Marker Lucifer yellow
Data Delivery Papp (A to B and B to A)
Efflux ratio

Available Caco-2 Permeability Assay at Creative Bioarray

  • Passive permeability in Caco-2
  • Evaluation of MDR1 substrate in Caco-2
  • Evaluation of BCRP substrate in Caco-2
  • Inhibitor assessment in Caco-2 (MDR1 and BCRP)

Caco-2 permeability assay is a part of our portfolio of in vitro ADME screening services. Creative Bioarray delivers consistent, high quality Caco-2 assay data with cost-efficiency that comes from a highly automated approach.

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