The Ussing Chamber Assay to Study Drug Metabolism and Transport in the Human Intestine

Beatrice Kisser1, Eva Mangelsen1, Caroline Wingolf2, Lars Ivo Partecke3, Claus‐Dieter Heidecke3, Christer Tannergren2, Stefan Oswald1, Markus Keiser1

1 Department of Clinical Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine Greifswald, Greifswald, 2 AstraZeneca R&D, Mölndal, Gothenburg, 3 Department of General Surgery, Visceral, Thoracic and Vascular Surgery, University Medicine Greifswald, Greifswald
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 7.17
DOI:  10.1002/cpph.22
Online Posting Date:  June, 2017
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Abstract

The Ussing chamber is an old but still powerful technique originally designed to study the vectorial transport of ions through frog skin. This technique is also used to investigate the transport of chemical agents through the intestinal barrier as well as drug metabolism in enterocytes, both of which are key determinants for the bioavailability of orally administered drugs. More contemporary model systems, such as Caco‐2 cell monolayers or stably transfected cells, are more limited in their use compared to the Ussing chamber because of differences in expression rates of transporter proteins and/or metabolizing enzymes. While there are limitations to the Ussing chamber assay, the use of human intestinal tissue remains the best laboratory test for characterizing the transport and metabolism of compounds following oral administration. Detailed in this unit is a step‐by‐step protocol for preparing human intestinal tissue, for designing Ussing chamber experiments, and for analyzing and interpreting the findings. © 2017 by John Wiley & Sons, Inc.

Keywords: ussing chamber; human intestine; in vitro; Papp‐value; transporter protein; metabolizing enzymes

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Determining Compound Transport Through Human Intestinal Tissue Using the Ussing Chamber Model
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Determining Compound Transport Through Human Intestinal Tissue Using the Ussing Chamber Model

  Materials
  • Agar‐salt bridges (see recipe)
  • Krebs‐bicarbonate Ringer's (KBR) solution (see recipe)
  • Custodiol HTK solution (e.g., Dr. Franz Köhler Chemie GmbH)
  • Saturated 3 M (224 g/liter) KCl solution (stable indefinitely at room temperature)
  • Carbogen gas (5% CO 2, 95% O 2)
  • Intestinal tissue
  • Substrate
  • Laminar flow box (e.g., Herasafe KS, Thermo Fisher Scientific, Darmstadt, Germany)
  • Syringe filters, PVDF, sterile (pore size 0.45 µm)
  • 20‐ or 50‐ml syringe
  • Two 250‐ml glass bottles
  • Styrofoam box filled with ice
  • Ussing chambers (Fig. ), including tissue clamp and tissue holder (Ussing chamber systems can be constructed in‐house or are commercially available from, e.g., Warner Instruments or World Precision Instruments)
  • Small thermometer to measure temperature inside the chambers
  • Circulation pumps (10°C for preparation bowl and 37°C for water jackets; e.g., Alpha A 6 and ECO RE 620 SWN; Lauda, Lauda‐Königshofen, Germany)
  • Two Ag‐AgCl electrodes/chamber (for voltage; e.g., Radiometer Analytical Reference Electrode REF321, Hach Lange GmbH, Düsseldorf, Germany)
  • Two silver (Ag) electrodes/chamber (for current; in‐house material made from silver wire)
  • Flexible tubing (15 to 17 cm; 4 mm i.d. × 6 mm o.d.)
  • Variable area flowmeters (e.g., V‐100 80, HTK, Hamburg, Germany)
  • Bowl with silicone mat for membrane preparation (not commercially available but must be a stainless steel bowl that can be cooled and gassed; Fig. )
  • Pressure reducing regulator (e.g., EN‐ISO 2503 for oxygen, GCE, Fulda, Germany)
  • Scissors (one button scissor or a straight Mayo scissor, One small sharp surgical scissors (e.g., Metzenbaum)
  • Two small and two long forceps
  • Fixing pins
  • Stereomicroscope (e.g., Leica M125, Wetzlar, Germany)
  • 10‐ml graduated cylinders
  • Water bath or incubator (e.g., AL25, Lauda, Lauda‐Königshofen, Germany)
  • Power supply if stirrers are used (e.g., PowerPac Universal Power Supply, BioRad)
  • Data acquisition and analysis system (e.g., UCC‐Labs 401 system, UCC‐Labs AB, Mölndal, Sweden)
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Figures

Videos

Literature Cited

Literature Cited
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