Micropatterned Co‐Cultures of Human Hepatocytes and Stromal Cells for the Assessment of Drug Clearance and Drug‐Drug Interactions

Christine Lin1, Salman R Khetani1

1 Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 14.17
DOI:  10.1002/cptx.23
Online Posting Date:  May, 2017
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Drug clearance rates from the body can determine drug exposure that can affect efficacy or toxicity. Thus, accurate prediction of drug clearance during preclinical development can help guide dose selection in humans, but animal testing is not always predictive of human outcomes. Because hepatic drug metabolism is a rate‐limiting step in the overall clearance of many drugs, primary human hepatocytes (PHHs) in suspension cultures or monolayers are used for drug clearance predictions. Yet, the precipitous decline in drug metabolism capacity can lead to significant underestimation of clearance rates, particularly for low turnover compounds that have desirable one‐pill‐a‐day dosing regimens. In contrast, micropatterned co‐cultures (MPCCs) of PHHs and fibroblasts display phenotypic stability for several weeks and can help mitigate the limitations of conventional cultures. Here, we describe protocols to create and use MPCCs for drug clearance predictions, and for modeling clinically‐relevant drug‐drug interactions that can affect drug clearance. © 2017 by John Wiley & Sons, Inc.

Keywords: primary human hepatocytes; cytochrome P450; 3T3‐J2 fibroblasts; low turnover drugs; drug metabolism

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Table of Contents

  • Introduction
  • Basic Protocol 1: Creation of Micropatterned Cocultures
  • Support Protocol 1: Constructing the PDMS Mask
  • Support Protocol 2: Culturing 3T3‐J2 Fibroblasts
  • Support Protocol 3: Assessing Hepatocyte Health/Functionality
  • Basic Protocol 2: Assessing Drug Clearance in MPCCs
  • Alternate Protocol 1: Assessing Drug‐Drug Interactions in MPCCs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Creation of Micropatterned Cocultures

  • Rat‐tail collagen type I (Corning, cat. no. 354236)
  • Sterile double‐distilled water (ddH 2O)
  • 70% (v/v) ethanol in ddH 2O
  • 0.05% (w/v) bovine serum albumin fraction V (Fisher Scientific, cat. no. BP1600‐100) in ddH 2O
  • Plateable cryopreserved PHHs (BioreclamationIVT, Triangle Research Laboratories, and Life Technologies)
  • Human hepatocyte seeding medium (see recipe)
  • Trypan blue
  • 1× Dulbecco's modified Eagle medium (DMEM), high‐glucose formulation
  • Human hepatocyte overnight medium (see recipe)
  • 3T3‐J2 fibroblasts (see protocol 3)
  • Human hepatocyte maintenance medium (see recipe)
  • Biosafety cabinet
  • 96‐well tissue‐culture plates, polystyrene
  • 37°C cell culture incubator
  • PDMS mask (see protocol 2)
  • Plate compression clamp (Star Prototype Manufacturing, Guangdong, China, or 3D Systems, Rockhill, SC)
  • Screwdriver
  • Oxygen tank (medical grade)
  • Oxygen plasma chamber (e.g., PlasmaEtch and SPI Supplies)
  • 37°C water bath
  • 50‐ml conical tubes
  • Hemocytometer
  • Serologic pipettes
  • Multichannel micropipettes with tips
  • Tissue culture microscope

Support Protocol 1: Constructing the PDMS Mask

  • Hexamethyldisilazane
  • Sylgard 184 PDMS kit (Dow Corning)
  • Silicon master wafer with SU‐8 photoresist (150‐250 µm thickness) negatively patterned in circular micro‐domains/islands of 500‐µm diameter that are spaced 1200 µm apart center‐to‐center (Trianja Technologies, SimTech, or FlowJem)
  • Glass petri dish
  • Weighing dishes
  • Vacuum desiccator
  • Arch punch (5‐mm diameter for a 96‐well plate)
  • Teflon blocks (e.g., Star Prototype Manufacturing, Guangdong, China, or 3D Systems, Rockhill, SC)
  • Oven

Support Protocol 2: Culturing 3T3‐J2 Fibroblasts

  • 3T3‐J2 murine embryonic fibroblasts (courtesy of Howard Green from Harvard Medical School)
  • Fibroblast medium (see recipe)
  • 1× phosphate buffered saline (PBS) solution
  • 0.25% (w/v) trypsin in 0.21 mm ethylenediaminetetraacetic acid (trypsin‐EDTA)
  • Tissue culture flasks (T‐150)

Support Protocol 3: Assessing Hepatocyte Health/Functionality

  • CYP450 luminescent kit (Promega)
  • CYP450 substrates reconstituted in dimethyl sulfoxide (DMSO)
  • Human hepatocyte dosing medium (see recipe)
  • Human hepatocyte maintenance medium (see recipe)
  • Human albumin ELISA kit (Bethyl Laboratories)
  • Urea nitrogen test kit (Stanbio Laboratory)
  • 96‐well collection plates
  • 96‐well assay plates
  • Multichannel micropipettes with tips
  • Spectrophotometer (ideally compatible with 96‐well plate for higher throughput)
  • Luminometer (ideally compatible with 96‐well plates for higher throughput)
  • Data analysis software (e.g., Microsoft Excel and GraphPad Prism)

Basic Protocol 2: Assessing Drug Clearance in MPCCs

  • Drugs of interest
  • DMSO
  • Human hepatocyte dosing medium (see recipe)
  • MPCCs seeded in 96‐well tissue culture plates (from protocol 1)
  • 1.5‐ml microcentrifuge tubes
  • 37°C water bath
  • 37°C incubator

Alternate Protocol 1: Assessing Drug‐Drug Interactions in MPCCs

  Additional Materials
  • Drugs of interest (perpetrator and victim drugs)
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