Use of Primary Rat Hepatocytes for Prediction of Drug‐Induced Mitochondrial Dysfunction

Cong Liu1, Shuichi Sekine1, Binbin Song1, Kousei Ito1

1 Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 14.16
DOI:  10.1002/cptx.24
Online Posting Date:  May, 2017
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Mitochondrial dysfunction plays a central role in drug‐induced liver injury. To evaluate drug‐induced mitochondrial impairment, several isolated mitochondria‐ or cell line‐based assays have been reported. Among them, culturing HepG2 cells in galactose provides a remarkable method to assess mitochondrial toxicity by activating mitochondrial aerobic respiration. We applied this assay to primary rat hepatocytes by culturing cells in galactose and hyperoxia to enhance the evaluation of metabolism‐related drug‐induced mitochondrial toxicity. Conventional culture of primary hepatocytes under high‐glucose and hypoxic conditions could force cells to switch energy generation to glycolysis. By contrast, cells cultured in galactose and hyperoxia could maintain energy generation from mitochondrial aerobic respiration, which is consistent with physiological conditions, and consequently improve the susceptibility of cells to mitochondrial toxicants. Measuring the toxicities of test compounds in primary rat hepatocytes cultured in modified conditions provides a useful model to identify mitochondrial dysfunction‐mediated drug‐induced hepatotoxicity. © 2017 by John Wiley & Sons, Inc.

Keywords: primary rat hepatocytes; mitochondrial toxicity; oxygen requirement; galactose; drug‐induced liver injury

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Table of Contents

  • Introduction
  • Basic Protocol 1: Sandwich Culture of Primary Rat Hepatocytes in Hyperoxic/Normoxic Conditions and Glucose/Galactose‐Based Medium
  • Basic Protocol 2: Treatment with Test Compounds and the LDH Assay in a 96‐Well Plate Format
  • Support Protocol 1: Isolation of Primary Rat Hepatocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Sandwich Culture of Primary Rat Hepatocytes in Hyperoxic/Normoxic Conditions and Glucose/Galactose‐Based Medium

  • Rat tail collagen type I (e.g., Corning)
  • 0.02 M acetic acid
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Freshly isolated rat hepatocytes (see Support Protocol 1)
  • Plating medium (see recipe)
  • Matrigel (e.g., Corning)
  • Glucose‐based culture medium (see recipe)
  • Galactose‐based culture medium (see recipe)
  • 96‐well tissue culture plate
  • Cell counter
  • Multi‐gas incubator (e.g., ASTEC‐Bio, APM‐30D)

Basic Protocol 2: Treatment with Test Compounds and the LDH Assay in a 96‐Well Plate Format

  • Test compounds
  • Dimethyl sulfoxide (DMSO)
  • All free glucose medium: William's E medium (no phenol red; e.g., Life Technologies, cat. no. A12176‐01)
  • All free galactose medium: Custom William's E medium (equivalent to Gibco cat. no. 12551‐032 except with no glucose; e.g., Cell Science & Technology) supplemented with 10 mM galactose
  • Apo‐transferrin, iron‐free, human (e.g., Nacalai Tesque)
  • Primary rat hepatocytes cultured in glucose/20% oxygen or galactose/80% oxygen (see protocol 1)
  • Triton X‐100 solution (at a final concentration of 0.25% [v/v])
  • Rotenone
  • Reaction solution: LDH cytotoxicity detection kit (e.g., TaKaRa Bio)
  • 1 M HCl
  • Multi‐gas incubator (e.g., ASTEC‐Bio, APM‐30D)
  • Centrifuge with plate adaptor
  • 96‐well tissue culture plate
  • Multiwell plate reader
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Literature Cited

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Internet Resources
  LDH Cytotoxicity Detection Kit user manuals.˜/media/files/resources/protocols/technical%20bulletins/0/celltiter%20glo%20luminescent%20cell%20viability%20assay%20protocol.pdf
  CellTiter‐Glo Luminescent Cell Viability Assay‐354234.pdf
  Corning Matrigel Basement Membrane Matrix.‐354236.pdf
  Corning Collagen 1, Rat Tail.
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