title: Optimizing Caco-2 cell monolayers to increase throughput in drug intestinal absorption analysis.
abstract: BACKGROUND
  The aim of this investigation was to evaluate methods for increasing Caco-2 cell throughput for assessing drug intestinal absorption. The use of 6-, 12-, and 24-well membranes and the effect of membrane size on permeability and the integrity of the Caco-2 cell monolayer were assessed. In an effort to optimize the assessment of drug permeability, increased throughput was investigated by testing compounds singly or as mixtures of analytes.
  
  
  METHODS
  The transepithelial electrical resistance (TEER) of cell monolayers was measured on 0.33, 1.0, and 4.7 cm2 polycarbonate membranes using EVOM, over a 25-day period. Absorptive transport was determined on all compounds tested using LC-MS/MS assays, or liquid scintillation spectrometry.
  
  
  RESULTS
  The effect of multiple compounds in one well compared to single compounds was assessed with atenolol, nadolol, metoprolol, and propranolol for mixtures of four compounds and with RWJ-53308, atenolol, terbutaline, propranolol, naproxen, piroxicam, topiramate, and furosemide for mixtures of eight compounds. The apparent permeability (Papp) values correlated well between single analytes and mixtures of four and eight analytes in each well. Drug permeability decreased slightly with an increase in well size. The TEER value increased with the number of days in culture for each of the 6-, 12-, and 24-well sizes.
  
  
  CONCLUSIONS
  It was demonstrated that the 24-well format system is ideal for high-throughput assessment. Furthermore, the approach of mixing four or eight analytes in each well to further increase throughput was also demonstrated to be valid.
authors.affiliation: Department of Drug Metabolism, The R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ 08869, USA. mmarkows@prius.jnj.com
authors.forename: M
authors.initials: M
authors.lastname: Markowska
authors.forename: R
authors.initials: R
authors.lastname: Oberle
authors.forename: S
authors.initials: S
authors.lastname: Juzwin
authors.forename: C P
authors.initials: CP
authors.lastname: Hsu
authors.forename: M
authors.initials: M
authors.lastname: Gryszkiewicz
authors.forename: A J
authors.initials: AJ
authors.lastname: Streeter
mesh_terms: D018938:Caco-2 Cells / Q000187:drug effects / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002853:Chromatography, Liquid; D004338:Drug Combinations; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D017097:Electric Impedance; D006801:Humans; D007408:Intestinal Absorption / Q000502:physiology*; D013058:Mass Spectrometry; D008567:Membranes, Artificial; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011076:Polycarboxylate Cement; D012588:Scintillation Counting
chemical_list: D004338:Drug Combinations; D008567:Membranes, Artificial; D004364:Pharmaceutical Preparations; D011076:Polycarboxylate Cement; C005506:polycarbonate