Development of a 7-day, 96-well Caco-2 permeability assay with high-throughput direct UV compound analysis

Pharm Res. 2003 Dec;20(12):1961-9. doi: 10.1023/b:pham.0000008043.71001.43.


Purpose: The aim was to replace the traditional 21-day Caco-2 permeability protocol by a more high-throughput assay.

Methods: Caco-2 cells were seeded at high density in 96-well plates in novel cell culture boxes. After 7 days, drug permeability studies were performed. Samples were analyzed by a new UV detection method.

Results: With increased cell seeding density. functional Caco-2 monolayers with polarized efflux transporters were established after 7 days in 96-well polycarbonate filter plates in standard medium. For faster feeding and to eliminate medium replacement in each individual well, plates were completely submerged in medium in novel cell culture boxes, and only medium outside the plate was exchanged. For high-throughput sample analysis, a novel UV-transparent transport buffer was established that allowed direct quantification of permeated drug from its UV absorption. In vitro permeability studies analyzing 22 passively absorbed drugs in the new model correlated well with reported human permeability values (r2 = 0.8725).

Conclusions: The new 7-day. 96-well Caco-2 permeability model tight to UV analysis offers considerable time, cost, and resource savings compared to the traditional model. It has a potential for automation and makes it possible to determine the permeability of passively diffusing compounds and to classify them according to the BCS in a truly medium- to high-throughput mode.

MeSH terms

  • Algorithms
  • Caco-2 Cells
  • Culture Media
  • Fluorescent Dyes
  • Humans
  • Hydrogen-Ion Concentration
  • Intestinal Absorption*
  • Isoquinolines
  • Pharmaceutical Preparations / metabolism
  • Reproducibility of Results
  • Spectrophotometry, Ultraviolet / methods*
  • Ultrafiltration


  • Culture Media
  • Fluorescent Dyes
  • Isoquinolines
  • Pharmaceutical Preparations
  • lucifer yellow