Caco‐2 Cells as a Model for Intestinal Absorption

Isabella De Angelis1, Laura Turco1

1 Istituto Superiore di Sanità, Rome, Italy
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 20.6
DOI:  10.1002/0471140856.tx2006s47
Online Posting Date:  February, 2011
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Abstract

The Caco‐2 cell system, a well characterized intestinal in vitro model, makes it possible to evaluate the ability of chemicals to cross the intestinal barrier, as well as to study their transport mechanisms. Permeability values estimated with this model correlate well with human in vivo absorption data for many drugs and chemicals. As a consequence, the use of the Caco‐2 cellular model as a permeability assay to predict oral absorption in humans is growing, and its importance is increasing as a screening tool in drug‐discovery strategies for the prediction of intestinal drug permeability. Strict observance of the experimental conditions, coupled with careful control of the cell‐culture procedures, are crucial for obtaining a meaningful correlation with in vivo data. Because of the intrinsic variability of this absorption model, it is important to highlight that each experimental system must be calibrated with internal reference compounds, to permit data sharing between different laboratories.Curr. Protoc. Toxicol. 47:20.6.1‐20.6.15. © 2011 by John Wiley & Sons, Inc.

Keywords: Caco‐2 cells; intestinal in vitro model; epithelial barrier; intestinal absorption; permeability studies; in vitro–in vivo correlations

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Methods for Caco‐2 Culture on Permeable Inserts
  • Basic Protocol 2: Measurement of Monolayer Integrity in the Presence of Test Compounds by Lucifer Yellow (LY) Passage
  • Alternate Protocol 1: Measurement of Monolayer Integrity in the Presence of Test Compounds by [14C]Mannitol Passage
  • Support Protocol 1: Preparation of Test Chemicals
  • Support Protocol 2: Transepithelial Electrical Resistance (TEER) Measurement
  • Support Protocol 3: Determination of Papp Value
  • Basic Protocol 3: Transport Experiments
  • Support Protocol 4: Analytical Procedures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Methods for Caco‐2 Culture on Permeable Inserts

  Materials
  • Caco‐2 cell culture medium (see recipe) with 10% and 20% FBS
  • Trypsin solution (TrypLE Select; Invitrogen)
  • Caco‐2 cell line (ATCC, cat. no. HTB‐37)
  • Cell culture microscope
  • Centrifuge
  • Cell culture insert: polyethylene terephthalate (PET) transparent membrane, 0.4‐ or 1‐µm pore size; 12‐well plates (BD Falcon or Millicell from Millipore)
  • Additional reagents and equipment for counting viable cells ( appendix 3B)

Basic Protocol 2: Measurement of Monolayer Integrity in the Presence of Test Compounds by Lucifer Yellow (LY) Passage

  Materials
  • 4.5 mM Lucifer Yellow stock solution (Sigma; prepare in sterile Milli‐Q water immediately before use)
  • Test compound stock solution in appropriate solvent ( protocol 4)
  • HBSS/MES assay buffer, pH 6.0 to 6.5 (see recipe)
  • HBSS/HEPES assay buffer, pH 7.2 to 7.4 (see recipe)
  • Caco‐2 cells growing on insert ( protocol 1)
  • Heating plate (Echo Term IC20, Torrey Pines Scientific, http://www.torreypinesscientific.com)
  • Circular plate shaker (IKA‐Schuettler MTS4)
  • Spectrofluorimeter
  • Additional reagents and equipment for preparing the test compound ( protocol 4), TEER measurements ( protocol 5), and calculation of Papp value ( protocol 6)

Alternate Protocol 1: Measurement of Monolayer Integrity in the Presence of Test Compounds by [14C]Mannitol Passage

  Materials
  • Mannitol D‐1 [14C] (CAS 69‐65‐8, specific activity 45 to 60 mCi (1.66 to 2.22 GBq)/mmol; PerkinElmer, NEC‐314)
  • HBSS/MES assay buffer, pH 6.0 to 6.5 (see recipe)
  • HBSS/ HEPES assay buffer, pH 7.2 to 7.4 (see recipe)
  • Test compound stock solution in appropriate solvent ( protocol 4)
  • Caco‐2 cells growing on insert ( protocol 1)
  • Liquid scintillation cocktail (Optiphase HiSafe; PerkinElmer)
  • Heating plate (Echo Term IC20, Torrey Pines Scientific)
  • Circular plate shaker (IKA‐Schuettler MTS4)
  • Scintillation vials
  • Scintillation counter (Beckman, LS‐6500)
  • Additional reagents and equipment for preparing the test compound ( protocol 4) and TEER measurements ( protocol 5)

Support Protocol 1: Preparation of Test Chemicals

  Materials
  • Test compound
  • Ultrapure sterile water (Milli‐Q‐purified water) for hydrophilic compounds or DMSO or ethanol for hydrophobic compounds
  • Dark glass vials with Teflon‐lined screw caps

Support Protocol 2: Transepithelial Electrical Resistance (TEER) Measurement

  Materials
  • 70% ethanol
  • Caco‐2 cell culture medium (see recipe) with 10% FBS
  • Heating plate (Echo Term IC20, Torrey Pines Scientific)
  • Chop‐stick electrodes device (Millicell‐ERS voltmeter, Millipore) or comparable device

Support Protocol 3: Determination of Papp Value

  Materials
  • Test compound stock solution in appropriate solvent ( protocol 4)
  • HBSS‐MES assay buffer, pH 6.0 to 6.5 (see recipe)
  • HBSS‐HEPES assay buffer pH 7.2 – 7.4 (see recipe)
  • Circular plate shaker (IKA‐Schuettler MTS4)
  • Heating plate (Echo Term IC20, Torrey Pines Scientific)
  • Glass dark vials with a screw cap with a Teflon membrane
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Figures

Videos

Literature Cited

Literature Cited
   Artursson, P. and Karlsson, J. 1991. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco‐2) cells. Biochem. Biophys. Res. Com. 175:880‐885.
   Artursson, P., Palm, K., and Luthman, K. 2001. Caco‐2 monolayers in experimental and theoretical predictions of drug transport. Adv. Drug Deliv. Rev. 46:27‐43.
   Cheng, K‐C., Li, C., and Uss, A.S. 2008. Prediction of oral drug absorption in humans from cultured cell lines and experimental animals. Exp. Opin. Drug Metab. Toxicol. 4:581‐590.
   FDA. 2000. Guidance for Industry: Waiver of in vivo Bioavailability and Bioequivalence Studies for Immediate‐Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System, U.S. Food and Drug Administration, Center for Drug Evaluation and Research, U.S.A. Available at: http://www.fda.gov/cder/guidance/3618fnl.pdf.
   Fogh J., Wright W.C., and Loveless, J.D. 1977. Absence of Hela cell contamination in 169 cell lines derived from human tumours. J. Natl. Cancer Inst. 21:393‐408.
   Gres, M.C., Julian, B., Bourrié, M., Meunier, V., Roques, C., Berger, M., Boulenc, X., Berger, Y., and Fabre, G. 1998. Correlation between oral drug absorption in humans, and apparent drug permeability in TC‐7 cells, a human epithelial intestinal cell line: Comparison with the parental Caco‐2 cell line. Pharm. Res. 15:726‐733.
   Hayeshi, R., Hilgendorf, C., Artursson, P., Augustijns, P., Brodin, B., Dehertogh, P., Fisher, K., Fossati, L., Hovenkamp, E., Korjamo, T., Masungi, C., Maubon, N., Mols, R., Müllertz, A., Mönkkönen, J., O'Driscoll, C., Oppers‐Tiemissen, H.M., Ragnarsson, E.G., Rooseboom, M., and Ungell, A.L. 2008. Comparison of drug transporter gene expression and functionality in Caco‐2 cells from 10 different laboratories. Eur. J. Pharm. Sci. 35:383‐396.
   Hilgendorf C., Ahlin G., Seithel A., Artursson P., Ungell A.L. and Karlsson J. 2007. Expression of thirty‐six drug transporter genes in human intestine, liver, kidney and organotypic cell lines. Drug Metab. Disp. 35:1333‐1340.
   Karlsson, J. and Artursson P. 1991. A method for the determination of cellular permeability coefficients and aqueous boundary layer thickness in monolayers of intestinal epithelial (Caco‐2) cells grown in permeable filter chambers. Int. J. Pharm. 71:55‐64.
   Neuhoff, S., Ungell, A.L., Zamora, I., and Artursson, P. 2005. pH‐dependent passive and active transport of acidic drugs across Caco‐2 cell monolayer. Eur. J. Pharm. Sci. 25:211‐220.
   Neutra, M. and Louvard, D. 1989. Differentiation of intestinal cells in vitro. In Functional Epithelial Cells in Culture (K.S. Mathin and J.D. Valurtich, eds.) pp. 363‐398. Alan R. Liss Inc., New York.
   Nigsch, F., Klaffe, W., and Miret, S. 2007. In vitro models for processes involved in intestinal absorption. Exp. Opin. Drug Metab. Toxicol. 3:545‐556.
   Pinto, M., Robine‐Leon, S., Appay, M.D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon‐Assmann, P., Haffen, K., Fogh, J., and Zweibaum, A. 1983. Enterocyte‐like differentiation and polarization of the human colon carcinoma cell line Caco‐2 in culture. Biol. Cell 47:323‐330.
   Prieto, P., Hoffmann, S., Tirelli, V., Tancredi, F., González, I., Bermejo, M., and De Angelis, I. 2010. An exploratory study of two Caco‐2 cell models for oral absorption: A report on their within‐laboratory and between‐laboratory variability, and their predictive capacity. ATLA 38:367‐386.
   Sambuy, Y., De Angelis, I., Ranaldi, G., Scarino, M.L., Stammati, A., and Zucco, F. 2005. The Caco‐2 cell line as a model of the intestinal barrier: Influence of cell and culture‐related factors on Caco‐2 cell functional characteristics. Cell Biol. and Toxicol. 21:1‐26.
   Sun, D., Hans Lennernas, H., Welage, L.S., Barnett, J.L, Landowski, C.P., Foster, D., Fleisher, D., Lee, K.D., and Amidon, G.L. 2002. Comparison of human duodenum and Caco‐2 gene expression profiles for 12,000 gene sequences tags and correlation with permeability of 26 drugs. Pharm. Res. 19:1400‐1416.
   Sun, H., Chow, E.CY., Liu, S., Du, Y., and Pang, K.S. 2008. The Caco‐2 cell monolayer: Usefulness and limitations. Exp. Opin. Drug Metab. Toxicol. 4:395‐411.
   Tavelin, S., Grasjo, J., Taipalensuu, J., Ocklind, G., and Artursson, P. 2002. Applications of epithelial cell culture in studies of drug transport. Methods Mol. Biol. 188:233‐272.
   Thomas, S., Brightman, F., Gill, H., Lee, S., and Pufong, B. 2007. Simulation modelling of human intestinal absorption using Caco‐2 permeability and kinetic solubility data for early drug discovery. J. Pharm. Sci. 97:4557‐4574.
   Turco, L., Catone, T., Caloni, F., Di Consiglio, E., Testai, E., and Stammati, A. 2011. Caco‐2/TC7 cell line characterization for intestinal absorption: How reliable is this in vitro model for the prediction of the oral dose fraction absorbed in human? Toxicol. in Vitro 25:13‐20.
   van Breemen, R.B. and Li, Y. 2005. Caco‐2 cell permeability assays to measure drug absorption. Exp. Opin. Drug Metab. Toxicol. 1:175‐185.
   Yamascita, S., Furubayashi, T., Kataoka, M., Sakane, T., Sezaki, H., and Tokuda, H. 2000. Optimized conditions for prediction of intestinal drug permeability using Caco‐2 cells. Eur. J. Pharm. Sci. 10:195‐204.
   Yee, S. 1997. In vitro permeability across Caco‐2 cells (colonic) can predict in vivo (small intestinal) absorption in man: Fact or myth. Pharm. Res. 14:763‐766.
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